Methods of treating metabolic disorders with dual function fibroblast growth factor 21 proteins

ABSTRACT

The present invention relates to the identification of dual function fusion proteins comprising fibroblast growth factor 21 (FGF21) and Exenatide, Exendin-4, or GLP-1. Also disclosed are methods for treating metabolic, cardiovascular, and endocrine conditions related to glucose and/or lipid homeostasis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 13/626,207 filed on Sep. 25, 2012, now U.S. Pat. No. 9,458,214, which claims the benefit of U.S. Provisional Application No. 61/539,290 filed on Sep. 26, 2011, each of which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 29, 2016, is named PAT054621-US-NP_SL.txt and is 343,718 bytes in size.

FIELD OF THE INVENTION

The present invention relates to new proteins comprising fibroblast growth factor 21 (FGF21) and other metabolic regulators known to improve metabolic profiles in subjects to whom they are administered.

BACKGROUND OF THE INVENTION

The fibroblast growth factor (FGF) family is characterized by 22 genetically distinct, homologous ligands, which are grouped into seven subfamilies. FGF-21 is most closely related to, and forms a subfamily with, FGF-19 and FGF-23. This FGF subfamily regulates diverse physiological processes uncommon to classical FGFs, namely energy and bile acid homeostasis, glucose and lipid metabolism, and phosphate as well as vitamin D homeostasis. Moreover, unlike other FGFs, this subfamily acts in an endocrine fashion (Moore, D. D. (2007) Science 316, 1436-8)(Beenken et al. (2009) Nature Reviews Drug Discovery 8, 235).

FGF21 is a 209 amino acid polypeptide containing a 28 amino acid leader sequence (SEQ ID NO:132). Human FGF21 has about 79% amino acid identity to mouse FGF21 and about 80% amino acid identity to rat FGF21. Fibroblast growth factor 21 (FGF21) has been described as a treatment for ischemic vascular disease, wound healing, and diseases associated with loss of pulmonary, bronchia or alveolar cell function (Nishimura et al. (2000) Biochimica et Biophysica Acta, 1492:203-206; patent publication WO01/36640; and patent publication WO01/18172). Although FGF-21 activates FGF receptors and downstream signaling molecules, including FRS2a and ERK, direct interaction of FGFRs and FGF-21 has not been detected. Studies have identified β-klotho, which is highly expressed in liver, adipocytes and pancreas, as a determinant of the cellular response to FGF-21 and a cofactor which mediates FGF-21 signaling through FGFRs (Kurosu, H. et al. (2007) J Biol Chem 282, 26687-95). FGF21 is a potent agonist of the FGFR1(IIIc), FGFR2(IIIc) and FGFR3(IIIc) β-klotho signaling complexes.

FGF-21 has been shown to induce insulin-independent glucose uptake. FGF-21 has also been shown to ameliorate hyperglycemia in a range of diabetic rodent models. In addition, transgenic mice over-expressing FGF-21 were found to be resistant to diet-induced metabolic abnormalities, and demonstrated decreased body weight and fat mass, and enhancements in insulin sensitivity (Badman, M. K. et al. (2007) Cell Metab 5, 426-37). Administration of FGF-21 to diabetic non-human primates caused a decline in fasting plasma glucose, triglycerides, insulin and glucagon levels, and led to significant improvements in lipoprotein profiles, including a nearly 80% increase in HDL cholesterol (Kharitonenkov, A. et al. (2007) Endocrinology 148, 774-81). Recent studies investigating the molecular mechanisms of FGF21 action have identified FGF21 as an important endocrine hormone that helps to control adaptation to the fasting state (Badman et al. (2009) Endocrinology 150, 4931)(Inagaki et al. (2007) Cell Metabolism 5, 415). This provides a previously missing link downstream of PPARa, by which the liver communicates with the rest of the body in regulating the biology of energy homeostasis (Galman et al. (2008) Cell Metabolism 8, 169)(Lundasen et al. (2007) Biochemical and Biophysical Research Communications 360, 437).

FGF21 regulates adipocyte homeostasis through activation of an AMPK/SIRT1/PGC1a pathway to inhibit PPARγ expression and increase mitochondrial function (Chau et al. (2010) PNAS 107, 12553). FGF21 also increases glucose uptake by skeletal muscle as measured in cultured human myotubes and isolated mouse tissue (Mashili et al. (2011) Diabetes Metab Res Rev 27, 286-97). FGF21 treatment of rodent islet cells leads to improved function and survival through activation of ERK1/2 and Akt pathways (Wente et al. (2006) Diabetes 55, 2470). FGF21 treatment also results in altered gene expression for lipogenesis and fatty acid oxidation enzymes in rodent livers, likely through HNF4a and Foxa2 signaling. However, recent studies (Wei et al. (2012) PNAS 109, 3143-48) indicate that treatment of diet-induced obese mice with FGF21 induces bone loss, due to a diminished inactivation of PPARγ (via reduced sumoylation); a shift of mesenchymal stem cell differentiation from osteoblasts to adipocytes is seen in the presence of increased PPARγ activity in the bone following FGF21 treatment.

A difficulty associated with using FGF-21 directly as a biotherapeutic is that its half-life is very short (Kharitonenkov, A. et al. (2005) Journal of Clinical Investigation 115:1627-1635). In mice, the half-life of human FGF21 is 0.5 to 1 hours, and in cynomolgus monkeys, the half-life is 2 to 3 hours. FGF21 may be utilized as a multi-use, sterile pharmaceutical formulation. However, it has been determined that preservatives, e.g., m-cresol, have an adverse effect on its stability under these conditions.

Another potent metabolic regulator already represented in the clinic is Glucagon-Like Peptide-1 (GLP-1) (Knudsen et al. (2004) Journal of Medicinal Chemistry 47, 4128). GLP-1 is a 36 amino acid incretin secreted by L-cells of the mammalian gut, acting on both alpha and beta cells to stimulate insulin secretion and inhibit glucagon release in a glucose-dependent manner (Hare et al. (2010) Diabetes 59, 1765; Meier et al. (2005) Diabetes-Metabolism Research and Reviews 21, 91). GLP-1 binds to and activates the GLP-1 receptor (GLP-1R), a seven-transmembrane helix protein of the class II family of G-protein coupled receptors (GPCRs) (Mayo et al. (2003) Pharmacological Reviews 55:167). As a GLP-1 receptor agonist, GLP-1 has an important role in decreasing post-prandial blood glucose levels by stimulating insulin secretion from the pancreas in order to increase glucose absorption in the peripheral tissues and inhibiting glucagon secretion, resulting in reduced hepatic glucose release.

A second clinically important GLP-1 receptor agonist is Exendin-4. Exendin-4 is a 39 residue polypeptide produced in the salivary glands of the Gila Monster lizard (Goke et al. (1993) Diabetes 46:433-439; Fehmann et al. (1995) Endocrine Rev. 16:390-410). Although it is the product of a uniquely non-mammalian gene and appears to be expressed only in the salivary gland, Exendin-4 shares a 52% amino acid sequence homology with GLP-1, and in mammals interacts with the GLP-1 receptor (Goke, et al.; Thorens et al. (1993) Diabetes 42:1678-1682). In vitro, Exendin-4 has been shown to promote insulin secretion by insulin producing cells and, given in equimolar quantities, is more potent than GLP-1 at causing insulin release from insulin producing cells. Furthermore, Exendin-4 potently stimulates insulin release to reduce plasma glucose levels in both rodents and humans and is longer acting than GLP-1; however, because it does not occur naturally in mammals, Exendin-4 has certain potential antigenic properties in mammals that GLP-1 lacks.

The ability of GLP-1 and Exendin-4 analogues (e.g., Liraglutide and Byetta) to improve glucose control in humans is established in the clinic (Idris (2010) Diabetes Obesity & Metabolism 12, 89; Monami et al (2009) European Journal of Endocrinology 160, 909). GLP-1 has also been reported to increase beta cell mass both through induced proliferation and inhibition of apoptosis (Egan, A et al (2003) Diabetes-Metabolism Research and Reviews 19, 115; Farilla, L. et al. (2003) Endocrinology 144, 5149; Xu, G. et al. (1999) Diabetes 48, 2270). It also acts as an intestinal hormone to inhibit acid secretion and gastric emptying in the stomach while providing a satiety signal that decreases appetite (Vilsboll et al. (2009) Best Practice & Research Clinical Endocrinology & Metabolism 23, 453). These effects likely account for beneficial weight loss observed with administration of GLP-1 analogues to type 2 diabetes patients. GLP-1 has also been shown to be cardioprotective in postischemic rodent hearts (Ossum et al. (2009) Pharmacological Research 60, 411; Sonne, D. P. et al. (2008) Regulatory Peptides 146, 243; Nikolaidis, L. A. et al. (2004) Circulation 109, 962).

Additionally, GLP-1 can reduce the differentiation of human mesenchymal stem cells (hMSCs) to adipocytes by reducing the expression of PPARγ, and GLP-1 promotes cellular proliferation and cytoprotection of hMSCs (Sanz et al. (2010) Am J Physiol Endocrinol Metab 298, E634-E643).

In developing an FGF21 protein, including a variant or analogue thereof, for use as a therapeutic in the treatment of type 1 and type 2 diabetes mellitus and other metabolic conditions, an increase in half-life and stability would be desirable. FGF21 proteins having enhanced half-life and stability would allow for less frequent dosing of patients being administered the protein. Clearly, there is a need to develop a stable aqueous protein formulation for the therapeutic protein FGF21.

Furthermore, a significant challenge in the development of protein pharmaceuticals, such as metabolic regulators FGF21, GLP-1, and Exendin-4, is to cope with their physical and chemical instabilities. The compositional variety and characteristics of proteins define specific behaviors such as folding, conformational stability, and unfolding/denaturation. Such characteristics should be addressed when aiming to stabilize proteins in the course of developing pharmaceutical formulation conditions utilizing aqueous protein solutions (Wang, W., Int. J. of Pharmaceutics, 18, (1999)). A desired effect of stabilizing therapeutic proteins of interest, e.g., the proteins of the present invention, is increasing resistance to proteolysis and enzymatic degradation, thereby improving protein stability and reducing protein aggregation.

SUMMARY OF THE INVENTION

The invention relates to the identification of new proteins, e.g., fusion proteins, which comprise fibroblast growth factor 21 (FGF21) and other metabolic regulators, e.g., GLP-1 and Exendin-4, and which have improved pharmaceutical properties over the constituent agents under pharmaceutical formulation conditions, e.g., are more stable, possess the ability to improve metabolic parameters for subjects to whom they are administered, are less susceptible to proteolysis and enzymatic degradation, are less likely to aggregate and form complexes and are less likely to be immunogenic. The proteins of the invention possess both FGF21 receptor agonist and GLP-1 receptor agonist activity; they comprise truncations and variants of FGF21, and further comprise one or more of, e.g., glucagon-like peptide-1 (GLP-1), Exendin-4, or other metabolic regulators or variants thereof.

Also disclosed are methods for treating FGF21-associated and GLP-1 associated disorders, as well as other metabolic, endocrine, and cardiovascular disorders, such as obesity, type 1 and type 2 diabetes mellitus, pancreatitis, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, acute myocardial infarction, hypertension, cardiovascular disease, atherosclerosis, peripheral arterial disease, stroke, heart failure, coronary heart disease, kidney disease, diabetic complications, neuropathy, gastroparesis, disorders associated with severe inactivating mutations in the insulin receptor, lipodystrophies including HIV-associated lypodistrophy and other metabolic disorders, and in reducing the mortality and morbidity of critically ill patients.

The proteins of the present invention may be used as a regularly administered (e.g., daily, more preferably weekly, biweekly, or monthly) injectable, either alone or in combination with oral anti-diabetic agents, which will improve the glycemic control, body weight and lipid profile of type 1 and type 2 diabetes mellitus patients. The proteins may also be used for the treatment of obesity or other FGF21- or GLP-1-associated conditions.

The proteins of the invention, e.g., GLP-1-FGF21 variant and Exendin-4-FGF21 variant fusion proteins of the invention, overcome the significant hurdles of physical instabilities associated with protein therapeutics, including, for instance, with the administration of the wild-type FGF21, as they are more stable, less susceptible to proteolysis and enzymatic degradation, less likely to aggregate and form complexes and less likely to be immunogenic than wild-type FGF21 under pharmaceutical formulation conditions.

In a first aspect, the invention provides Fibroblast Growth Factor 21 (FGF21) proteins, e.g., fusion proteins, comprising one or more of the sequences listed in Table 1, and further described herein. The proteins of the invention can further comprise GLP-1 and/or Exendin-4 proteins, whether wild-type, truncated, or mutated versions, or variants thereof. The FGF21 sequences listed in Table 1 are variants of the wild-type FGF21 sequence, e.g., the wild-type FGF21 sequence with NCBI reference number NP_061986.1, and found in such issued patents as, e.g., U.S. Pat. No. 6,716,626B1, assigned to Chiron Corporation. The GLP-1 and Exendin-4 sequences listed in Table 1 are variants of the wild-type GLP-1 and Exendin-4 sequences, e.g., those sequences with NCBI reference numbers NP_002045 and AAB22006.1, respectively, and can be found in such patent publications as, e.g., WO98/19698 and WO87/06941A, assigned to Eli Lilly and Co. and the General Hospital Corp., respectively (GLP-1) and U.S. Pat. No. 5,424,286, assigned to Amylin (Exendin-4).

Other embodiments are drawn to polynucleotides encoding the dual function proteins of the invention, a vector containing said polynucleotides and a host cell carrying said vector.

Provided herein are methods used to generate the proteins of the invention, wherein such methods involve modification of the wild-type FGF21 protein, via, e.g., the site-specific incorporation of amino acids at positions of interest within the wild-type FGF21 protein, as well as the fusion between the FGF21 portion of the molecule to other metabolic regulators, such as glucagon-like peptide-1 (GLP-1) and Exendin-4, or conjugates with polymers modified with GLP-1 and/or Exendin-4. Said modifications and fusions enhance the biological properties of the proteins of the invention relative to the wild-type versions of the proteins (e.g., FGF21, GLP-1, and Exendin-4), as well as, in some cases, serving as points of attachment for, e.g., labels and protein half-life extension agents, and for purposes of affixing said variants to the surface of a solid support. Related embodiments of the invention are methods to produce cells capable of producing said proteins of the invention, and of producing vectors containing DNA encoding said variants and fusions.

In various embodiments, the proteins of the invention can comprise one or more fragments of the FGF21, Exendin-4, and/or GLP-1 sequences, including fragments as small as 8-12 amino acid residues in length, wherein the polypeptide is capable of lowering blood glucose in a mammal. In various embodiments, the proteins of the invention can comprise one or more variants of the FGF21, Exendin-4, and/or GLP-1 sequences, e.g., with one or more amino acid deletions, insertions, additions, or substitutions relative to the wild-type sequences thereof.

In some embodiments, the proteins of the invention can be covalently linked to one or more polymers, such as polyethylene glycol (PEG) or polysialic acid, whether at the position of site-specific amino acid modifications made relative to the wild-type FGF21, GLP-1, or Exendin-4, or at the position of amino acids commonly shared with the wild-type versions of those proteins. The PEG group is attached in such a way so as enhance, and/or not to interfere with, the biological function of the constituent portions of the fusion proteins of the invention, e.g., the GLP-1 protein variants or FGF21 protein variants. In other embodiments, the polypeptides of the invention can be fused to a heterologous amino acid sequence, optionally via a linker, such as GS, GGGGSGGGGSGGGGS (SEQ ID NO:8), or SGGGGSGGG (SEQ ID NO:128). The heterologous amino acid sequence can be an IgG constant domain or fragment thereof (e.g., the Fc region), Human Serum Albumin (HSA), or albumin-binding polypeptides. Such fusion proteins disclosed herein can also form multimers.

In some embodiments, a heterologous amino acid sequence (e.g., HSA, Fc, etc.) is fused to the amino-terminal of the proteins of the invention. In other embodiments, the fusion heterologous amino acid sequence (e.g., HSA, Fc, etc.) is fused to the carboxyl-terminal of the proteins of the invention. In still other, more preferred embodiments, the heterologous amino acid sequence (e.g., HSA, Fc, etc.) is situated in the middle of the dual function proteins of the invention, i.e., between the C-terminal residue of the GLP-1 or Exendin-4 sequence and the N-terminal residue of the FGF21 sequence. Said preferred embodiment, e.g., leaves a free N-terminus for maximum GLP-1 (Exendin-4) activity and a free, intact C-terminus for maximum FGF21 activity.

In some embodiments, the GLP-1 receptor agonist is fused to the N-terminus of heavy and light chain of an antibody and FGF21 is simultaneously fused to the C-terminus of heavy and light chain of the same antibody (i.e., a fusobody, as described herein). Said preferred embodiment leaves a free N-terminus for maximum GLP-1 (Exendin-4) activity and a free, intact C-terminus for maximum FGF21 activity. A preferred embodiment uses the antibody sequence described in PCT publication WO2011/076781.

In some embodiments, the GLP-1 or Exendin-4 peptide is chemically attached to FGF21. In some embodiments, said peptides are attached to an FGF21 amino acid residue side chain. In other embodiments, said peptides are attached to the N-terminus of FGF21 through native chemical ligation or other methods known to the art. The preferred embodiment leaves a free N-terminus of GLP-1 (Exendin-4) for maximal activity and a free, intact C-terminus of FGF21 for maximal activity.

In some embodiments, the GLP-1 or Exendin-4 peptide is covalently attached to a polymer molecule that in turn is attached to the FGF21 protein variant. In a preferred embodiment, the GLP-1 or Exendin-4 peptide is attached to a PEG polymer that is simultaneously attached to a FGF21 protein variant. Said preferred embodiment leaves a free N-terminus for maximum GLP-1 (Exendin-4) activity and a free, intact C-terminus for maximum FGF21 activity. In some embodiments of the invention, a GLP-1 receptor agonist peptide is connected to a FGF21 variant through a PEG linker or other polymer linker that simultaneously provides half-life extension as well as a covalent connection for the two receptor agonists.

Yet another embodiment is drawn to methods of treating a patient exhibiting one or more FGF21-associated disorders or GLP-1-associated disorders, such as obesity, type 2 diabetes mellitus, type 1 diabetes mellitus, pancreatitis, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, acute myocardial infarction, hypertension, cardiovascular disease, atherosclerosis, peripheral arterial disease, stroke, heart failure, coronary heart disease, kidney disease, diabetic complications, neuropathy, gastroparesis, disorders associated with inactivating mutations in the insulin receptor, lipodystrophies including HIV-associated lipodystrophy and other metabolic disorders, comprising administering to said patient in need of such treatment a therapeutically effective amount of one or more proteins of the invention or a pharmaceutical composition thereof.

The invention also provides pharmaceutical compositions comprising the dual function proteins of the invention disclosed herein and a pharmaceutically acceptable formulation agent. Such pharmaceutical compositions can be used in a method for treating a metabolic disorder, and the method comprises administering to a human patient in need thereof a pharmaceutical composition of the invention. Non-limiting examples of metabolic disorders that can be treated include type 1 and type 2 diabetes mellitus and obesity.

These and other aspects of the invention will be elucidated in the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the activity of GLP-1-FGF21-PEG fusion proteins with different N-terminal mutations, and Exendin-4-FGF21-PEG fusion proteins. FIG. 1A shows the mutations added to slow processing by DPP-4 protease (GLP-1 peptide (circles) or dual function proteins with wild-type (V231; open squares), GO (V251; open circles), A8G (V258; open triangles), A8S (V232; triangles), or E9P (V271; squares) GLP-1). FIG. 1B shows Exendin-4 fusions (GLP-1 peptide (squares) or dual function proteins with Exendin-4 1-39 (V234; triangles), 1-30 (V267; circles) or 1-30/E16G/E17Q (V268; open triangles)).

FIG. 2 shows pharmacokinetic properties (PK) of the GLP-1-FGF21-PEG fusion proteins (FGF21-PEG (V294; open squares), GLP-1(A8S)-PEG (V253; open circles) or dual function proteins with wild-type GLP-1 (V237; circles) or GLP-1(A8S) (V235; triangles) GLP-1). The PK of wild-type non-PEGylated FGF21 injected at 0.25 mg/kg is shown for comparison (squares).

FIGS. 3A-3C show results from using the oral glucose tolerance test (OGTT) to measure the efficacy of half-life extended GLP-1-FGF21-PEG fusion proteins. Eight week C57BL/6J mice (n=5) were dosed by intraperitoneal (i.p.) injection with 1 mg/kg compound or vehicle (solid white bars; triangles). Blood glucose was measured at 1 and 24 hours post dose to assess acute effects of the GLP-1, which were similar for the wild-type GLP-1 (V239; hashed bars; squares) and GLP-1(A8S) versions (V232; black bars; circles) at 1 hour and retained slightly better by the GLP-1(A8S) version at 24 hours. On the third night, the mice were fasted before challenge with 1.5 g/kg oral glucose at 72 hours post-dose. The mice dosed with the A8S version showed significantly improved control of blood glucose compared to those with the wild-type GLP-1 version, suggesting that the A8S mutation increased long-term levels of active GLP-1.

FIG. 4 shows efficacy of GLP-1-FGF21-PEG fusion proteins in ob/ob mice. Ten week old male ob/ob mice (n=8) were dosed i.p. twice weekly with the indicated compounds for two weeks. Equivalent dosing of FGF21-PEG (V238; diagonal hashed bars; squares) and GLP-1-FGF21(R154C)-PEG (V239; horizontal hashed bars; open squares) showed very similar improvements in blood glucose levels, body weight, and liver health (as measured by serum ALT levels and liver weight) when compared to the vehicle-treated group (circles). The DPP-4-resistant fusion, 0.2 mg/kg GLP-1(A8S)-FGF21(R154C)-PEG (V235; vertical hashed bars; open triangles) showed similar efficacy to other compounds, but equal dosing at 1 mg/kg GLP-1(A8S)-FGF21(R154C)-PEG (V235; black bars; triangles) gave additional lowering of blood glucose, body weight, alanine aminotransferase (ALT), and liver weight.

FIGS. 5A-5G show a comparison of the GLP-1(A8S)-FGF21(R154C)-PEG (V235; black bars; open triangles) fusion protein to co-administration of FGF21(R154C)-PEG+GLP-1(A8S)-PEG (vertical hashed bars; open circles), single administration of FGF21(R154C)-PEG (V238; 0.2 mg/kg (light diagonal hashed bars); 1 mg/kg (dark diagonal hashed bars; squares)), and single administration of GLP-1(A8S)-PEG (V253; 0.2 mg/kg (light horizontal hashed bars); 1 mg/kg (dark horizontal hashed bars; open squares)) in ob/ob mice. Nine week old male ob/ob mice (n=8) were dosed i.p. twice weekly with the indicated compounds or vehicle (solid white bars; circles) for four weeks. The fusion showed significantly improved efficacy compared to co-administration of FGF21(R154C)-PEG+GLP-1(A8S)-PEG. Although only moderate weight loss was observed with FGF21(R154C)-PEG and GLP-1(A8S)-FGF21(R154C)-PEG, other groups gained a substantial amount of weight, only in part due to differences in food intake.

FIGS. 6A-6F show efficacy of GLP-1(A8S)-FGF21-PEG fusion proteins in ob/ob mice, comparing FGF21(R154C)-PEG (V235; dark bars) to FGF21(V76)-PEG (V272; light bars). Nine week old male ob/ob mice (n=8) were dosed i.p. twice weekly with the indicated compounds or vehicle (solid white bars) for two weeks. Both the GLP-1(A8S)-FGF21(R154C)-PEG and the GLP-1(A8S)-FGF21(V76)-PEG fusions showed a dose response from 0.05 to 0.2 mg/kg (0.05 diagonal hashed bars; 0.1 horizontal hashed bars; 0.2 vertical hashed bars) for glucose control and body weight. At 0.2 mg/kg, GLP-1(A8S)-FGF21(V76)-PEG was more efficacious than GLP-1(A8S)-FGF21FGF21(R154C)-PEG particularly for lowering of glucose levels, body weight, serum triglycerides and cholesterol. FIGS. 6A-6C show levels of basal glucose (AUC), alanine aminotransferase (ALT), and serum total cholesterol, respectively. FIGS. 6D-6F show levels of D12 body weight, hepatic lipids, and serum triglycerides, respectively.

FIGS. 7A-7B show the ability of GLP-1(A8S)-FGF21(V76)-PEG fusion proteins to improve pancreatic function and increase islet insulin content, relative to the combination of the individual agents. Db/db mice were dosed twice per week, for four weeks, with a combination of individual GLP-1(A8S)-PEG+FGF21(V76)-PEG (V76+V253; vertical hashed bars), as well as with the GLP-1(A8S)-FGF21(V76)-PEG dual function fusion protein of the invention (V272; diagonal hashed bars; light for 0.5 mg/kg and dark for 1 mg/kg), or vehicle (white bars).

FIGS. 8A-8B show the ability of GLP-1-FGF21-PEG fusion proteins to improve glucose lowering (FIG. 8A) and body weight (FIG. 8B), relative to the combination of the individual agents. Db/db mice were dosed twice per week, for two weeks, with vehicle (white bars; open circles), a combination of individual GLP-1(A8S)-PEG (V253; 3 mg/kg (light horizontal hashed bars; open diamonds) and 5 mg/kg (dark horizontal bars; diamonds)) and FGF21(V76)-PEG (3 mg/kg (V76; light diagonal hashed bars; open squares) and 5 mg/kg (dark diagonal hashed bars; squares)), as well as with the GLP-1(A8S)-FGF21(V76)-PEG dual function fusion protein of the invention (V272; 0.2 mg/kg (smelled checked bars; open triangles); 1 mg/kg (large checked bars; triangles)). As seen in the figure, 0.2 mg/kg of the GLP-1(7-35; A8S)-FGF21(V76)-PEG dual function fusion protein (V272) is as effective as 5 mg/kg of FGF21(V76)-PEG. Also as seen in the figure, 1.0 mg/kg of the GLP-1(7-35; A8S)-FGF21(V76)-PEG dual function fusion protein (V272) is more effective that the maximal effective combination doses of FGF21(V76)-PEG+GLP-1(7-35; A8S)-PEG (1+1 mg/kg (light horizontal hashed bars; exes) and 3+3 mg/kg (dark horizontal bars; stars), for both glycemic and body weight endpoints.

FIG. 9 shows the results of an assay for FGF21 activity. Phosphorylation of ERK is measured after treatment of HEK293-beta-klotho cells with the indicated compounds. The activity of GLP-1(A8S)-V76-PEG (V272) is significantly higher in magnitude of signal compared to the same FGF21 variant FGF21(V76)-PEG in the presence or absence of equimolar Exenatide.

FIGS. 10A-D present results of a receptor pharmacology assay used to measure the activity of fusion proteins compared to matched single agonist proteins or peptides. HEK293 cells transfected with GLP-1R and FGFR1c/beta-klotho (FIGS. 10A and 10B) or with GLP-1R alone (FIGS. 10C and 10D) were assayed for beta-arrestin recruitment to GLP-1R after treatment with compounds for 1 hour. Molecules tested in the assay were: Exendin-4 (squares), GLP-1(A8S)-FGF21(V76)-PEG (V272; circles), GLP-1(A8S)-PEG (V253; triangles), V253+FGF21(V76)-PEG (open diamonds), Exendin-4(1-39)-Fc-FGF21(V103) (V211; open circles), Exendin-4(1-39)-Fc (V201; open triangles), and V201+FGF21(V101) (diamonds).

DETAILED DESCRIPTION OF THE INVENTION

The proteins of the present invention represent modified versions of the full-length, wild-type FGF21 polypeptide, as known in the art. FGF21 wild-type sequence will serve as a reference sequence (SEQ ID NO:1), for instance, when comparisons between the FGF21 wild-type sequence and the protein variants are necessary. The FGF21 wild-type sequence has NCBI reference sequence number NP_061986.1, and can be found in such issued patents as, e.g., U.S. Pat. No. 6,716,626B1, assigned to Chiron Corporation (SEQ ID NO:1).

Met Asp Ser Asp Glu Thr Gly Phe Glu His Ser Gly Leu Trp Val Ser 1               5                   10                  15 Val Leu Ala Gly Leu Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro             20                  25                  30 Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr         35                  40                  45 Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg     50                 55                  60 Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu 65                  70                  75                  80 Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val                 85                  90                  95 Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly             100                 105                 110 Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu         115                 120                 125 Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu     130                 135                 140 His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly 145                 150                 155                 160 Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu                 165                 170                 175 Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp             180                 185                 190 Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala         195                 200                 205 Ser 209

The corresponding mRNA sequence coding for the full-length FGF21 polypeptide (NCBI reference sequence number NM_019113.2) is shown below (SEQ ID NO:2):

  1 ctgtcagctg aggatccagc cgaaagagga gccaggcact caggccacct gagtctactc  61 acctggacaa ctggaatctg gcaccaattc taaaccactc agcttctccg agctcacacc 121 ccggagatca cctgaggacc cgagccattg atggactcgg acgagaccgg gttcgagcac 181 tcaggactgt gggtttctgt gctggctggt cttctgctgg gagcctgcca ggcacacccc 241 atccctgact ccagtcctct cctgcaattc gggggccaag tccggcagcg gtacctctac 301 acagatgatg cccagcagac agaagcccac ctggagatca gggaggatgg gacggtgggg 361 ggcgctgctg accagagccc cgaaagtctc ctgcagctga aagccttgaa gccgggagtt 421 attcaaatct tgggagtcaa gacatccagg ttcctgtgcc agcggccaga tggggccctg 481 tatggatcgc tccactttga ccctgaggcc tgcagcttcc gggagctgct tcttgaggac 541 ggatacaatg tttaccagtc cgaagcccac ggcctcccgc tgcacctgcc agggaacaag 601 tccccacacc gggaccctgc accccgagga ccagctcgct tcctgccact accaggcctg 661 ccccccgcac tcccggagcc acccggaatc ctggcccccc agccccccga tgtgggctcc 721 tcggaccctc tgagcatggt gggaccttcc cagggccgaa gccccagcta cgcttcctga 781 agccagaggc tgtttactat gacatctcct ctttatttat taggttattt atcttattta 841 tttttttatt tttcttactt gagataataa agagttccag aggagaaaaa  aaaaaaaaa 901 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

The mature FGF21 sequence lacks a leader sequence and may also include other modifications of a polypeptide such as proteolytic processing of the amino terminus (with or without a leader sequence) and/or the carboxyl terminus, cleavage of a smaller polypeptide from a larger precursor, N-linked and/or O-linked glycosylation, and other post-translational modifications understood by those with skill in the art. A representative example of a mature FGF21 sequence has the following sequence (SEQ ID NO:3, which represents amino acid positions 29-209 of full-length FGF21 protein sequence (NCBI reference sequence number NP_061986.1)):

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val                  5                  10                  15  Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His              20                  25                  30  Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser          35                  40                  45  Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln      50                  55                  60 Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly  65                  70                  75                  80 Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg                  85                  90                  95 Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His             100                 105                 110 Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro         115                 120                 125 Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro     130                 135                 140 Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145                 150                 155                 160 Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser                 165                 170                 175 Pro Ser Tyr Ala Ser             180

The corresponding cDNA sequence coding for the mature FGF21 polypeptide (SEQ ID NO:3) is shown below (SEQ ID NO:4):

  1 caccccatcc ctgactccag tcctctcctg caattcgggg gccaagtccg gcagcggtac  61 ctctacacag atgatgccca gcagacagaa gcccacctgg agatcaggga ggatgggacg 121 gtggggggcg ctgctgacca gagccccgaa agtctcctgc agctgaaagc cttgaagccg 181 ggagttattc aaatcttggg agtcaagaca tccaggttcc tgtgccagcg gccagatggg 240 gccctgtatg gatcgctcca ctttgaccct gaggcctgca gcttccggga gctgcttctt 301 gaggacggat acaatgttta ccagtccgaa gcccacggcc tcccgctgca cctgccaggg 360 aacaagtccc cacaccggga ccctgcaccc cgaggaccag ctcgcttcct gccactacca 421 ggcctgcccc ccgcactccc ggagccaccc ggaatcctgg ccccccagcc ccccgatgtg 481 ggctcctcgg accctctgag catggtggga ccttcccagg gccgaagccc cagctacgct 541 tcctga

The proteins of the invention of the present invention represent modified versions of the full-length, wild-type GLP-1 polypeptide, as known in the art. GLP-1 wild-type sequence will serve as a reference sequence (SEQ ID NO:5), for instance, when comparisons between the GLP-1 wild-type sequence and the protein variants are necessary.

The GLP-1 wild-type sequence is post-translationally modified, and otherwise derived from, preproglucagon wild-type sequence. Preproglucagon sequence (SEQ ID NO:5) has NCBI reference sequence number NP_002045, and can be found in such patent publications as, e.g., WO98/19698 and WO87/06941A, assigned to Eli Lilly and Co. and the General Hospital Corp., respectively. As described, for example, in Goke, et al. (1991) European Journal of Clinical Investigation 21, 135, GLP-1 is a 37mer that is derived from preproglucagon (GLP-1 constitutes residues 92-138 of preproglucagon, and is underlined in SEQ ID NO:5, below). GLP-1 is further processed into an active 31mer, by cleavage of the six N-term residues.

  1 MKSIYFVAGL FVMLVQGSWQ RSLQDTEEKS RSFSASQADP LSDPDQMNED KRHSQGTFTS  61 DYSKYLDSRR AQDFVQWLMN TKRNRNNIAK RHDEFERHAE GTFTSDVSSY LEGQAAKEFI 121 AWLVKGRGRR DFPEEVAIVE ELGRRHADGS FSDEMNTILD NLAARDFINW LIQTKITDRK

An example of the processed, wild-type GLP-1 sequence is as follows (SEQ ID NO:129):

1 HDEFERHAEG TFTSDVSSYL EGQAAKEFIA WLVKGRG

An example of the active GLP-1 sequence is as follows (SEQ ID NO:30):

1 HAEGTFTSDV SSYLEGQAAK EFIAWLVKGR G

The corresponding mRNA sequence coding for the preproglucagon GLP-1 wild-type sequence (SEQ ID NO:5) is shown below (SEQ ID NO:6):

   1 gcatagaatg cagatgagca aagtgagtgg gagagggaag tcatttgtaa caaaaactca   61 ttatttacag atgagaaatt tatattgtca gcgtaatatc tgtgaggcta aacagagctg  121 gagagtatat aaaagcagtg cgccttggtg cagaagtaca gagcttagga cacagagcac  181 atcaaaagtt cccaaagagg gcttgctctc tcttcacctg ctctgttcta cagcacacta  241 ccagaagaca gcagaaatga aaagcattta ctttgtggct ggattatttg taatgctggt  301 acaaggcagc tggcaacgtt cccttcaaga cacagaggag aaatccagat cattctcagc  361 ttcccaggca gacccactca gtgatcctga tcagatgaac gaggacaagc gccattcaca  421 gggcacattc accagtgact acagcaagta tctggactcc aggcgtgccc aagattttgt  481 gcagtggttg atgaatacca agaggaacag gaataacatt gccaaacgtc acgatgaatt  541 tgagagacat gctgaaggga cctttaccag tgatgtaagt tcttatttgg aaggccaagc  601 tgccaaggaa ttcattgctt ggctggtgaa aggccgagga aggcgagatt tcccagaaga  661 ggtcgccatt gttgaagaac ttggccgcag acatgctgat ggttctttct ctgatgagat  721 gaacaccatt cttgataatc ttgccgccag ggactttata aactggttga ttcagaccaa  781 aatcactgac aggaaataac tatatcacta ttcaagatca tcttcacaac atcacctgct  841 agccacgtgg gatgtttgaa atgttaagtc ctgtaaattt aagaggtgta ttctgaggcc  901 acattgcttt gcatgccaat aaataaattt tcttttagtg ttgtgtagcc aaaaattaca  961 aatggaataa agttttatca aaatattgct aaaatatcag ctttaaaata tgaaagtgct 1021 agattctgtt attttcttct tattttggat gaagtacccc aacctgttta catttagcga 1081 taaaattatt tttctatgat ataatttgta aatgtaaatt attccgatct gacatatctg 1141 cattataata ataggagaat agaagaactg gtagccacag tggtgaaatt ggaaagagaa 1201 ctttcttcct gaaacctttg tcttaaaaat actcagcttt caatgtatca aagatacaat 1261 taaataaaat tttcaagctt ctttaccaaa aaaaaaaa

The proteins of the invention of the present invention represent modified versions of the full-length, wild-type Exendin-4 polypeptide, as known in the art. Exendin-4 wild-type sequence will serve as a reference sequence (SEQ ID NO:7), for instance, when comparisons between the Exendin-4 wild-type sequence and the protein variants are necessary.

The Exendin-4 wild-type sequence has NCBI reference sequence number GenBank: AAB22006.1, and can be found in such issued patents as, e.g., U.S. Pat. No. 5,424,286, assigned to Amylin Pharmaceuticals, Inc. and Eli Lilly and Co. An example of the wild-type sequence is as follows (SEQ ID NO:7):

1 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS

The proteins of the invention may comprise protein variants or mutants of the wild-type proteins listed herein, e.g., FGF21 variants, GLP-1 variants, and/or Exendin-4 variants. As used herein, the terms “protein variant,” “human variant,” “polypeptide or protein variant,” “variant,” “mutant,” as well as any like terms or specific versions thereof (e.g., “FGF21 protein variant,” “human GLP-1 variant,” “Exendin-4 polypeptide or protein variant,” “variant,” “FGF21 mutant,” etc.) define protein or polypeptide sequences that comprise modifications, truncations, other variants of naturally occurring (i.e., wild-type) protein or polypeptide counterparts or corresponding native sequences. “Variant FGF21” or “FGF21 mutant,” for instance, is described relative to the wild-type (i.e., naturally occurring) FGF21 protein as described herein.

Representative dual function protein sequences of the invention are listed in Table 1. The descriptions of said agonists include the individual constituent agonists and, where applicable a linker. If a variant is used as a constituent agonist, the changes or substitutions made are numbered relative to their wild-type counterparts. By way of example, “Dual Function 1—Protein” (SEQ ID NO:9) contains residues 7-35 of the wild-type GLP-1 sequence as described herein (i.e., a GLP-1 receptor agonist), a linker sequence, and an FGF21 variant (i.e., an FGF21 receptor agonist) with a number of listed changes to the FGF21 wild-type sequence (SEQ ID NO:1) as described herein.

TABLE 1  Dual Function Protein and Nucleotides of the invention SEQ ID Variant Short NO: NO: Sequence Long Name Name 9 V272 HSEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7- GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD 35; A8S)- 1(A8S)- AQETEAHLEI REDGTVGGAA HQSPESLLEL GSGGGGSGGG- L10- KALKPGVIQI LGVKTSRFLC QKPDGALYGS FGF21(33- FGF21 LHFDPEACSF RELLLEDGYN VYQSEAHGLP 209; Q56E- (V76)- LHLPGNRSPH CDPAPQGPAR FLPLPGLPPA D74H-Q82E- 40KPEGb LPEPPGILAP QPPDVGSSDP LAMVGPSQGR R105K- SPSYAS K150R- R154C-40 kDa branched PEG-R159Q- 5195A) 10 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7- GLP- TCTAGCTACC TGGAAGGCCA GGCTGCGAAA 35; A8S)- 1(A8S)- GAATTCATCG CGTGGCTGGT TAAAGGCGGT GSGGGGSGGG- L10- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAT FGF21(33- FGF21 AGCAGCCCGC TGCTGCAGTT TGGCGGCCAG 209; Q56E- (V76) GTGCGTCAGC GTTATCTGTA TACCGATGAT D74H-Q82E- GCGCAGGAAA CCGAAGCGCA TCTGGAAATT R105K- CGTGAAGATG GCACCGTGGG CGGTGCGGCG K150R- CATCAGAGCC CGGAAAGCCT GCTGGAACTG R154C- AAAGCGCTGA AACCGGGCGT GATTCAGATT R159Q- CTGGGCGTGA AAACCAGCCG TTTTCTGTGC 5195A) CAGAAACCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACCG TAGCCCGCAT TGCGATCCGG CACCGCAGGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGGCGATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 11 V277 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1- SGGGGSGGGG SGGGGSGGGG DSSPLLQFGG 30)- 30)- QVRQRYLYTD DAQETEAHLE IREDGTVGGA SGGGGSGGGGS L20- AHQSPESLLE LKALKPGVIQ ILGVKTSRFL GGGGSGGGG- FGF21 CQKPDGALYG SLHFDPEACS FRELLLEDGY FGF21(33- (V76)- NVYQSEAHGL PLHLPGNRSP HCDPAPQGPA 209; Q56E- 40KPEGb RFLPLPGLPP ALPEPPGILA PQPPDVGSSD D74H-Q82E- PLAMVGPSQG R105K- RSPSYAS K150R- R154C-40 kDa branched PEG-R159Q- 5195A) 12 CATGGTGAGG GTACGTTTAC TTCTGATCTG Exendin4(1- Ex(1- TCTAAACAGA TGGAAGAAGA AGCTGTTCGC 30)- 30)- CTGTTCATTG AATGGCTGAA AAATGGTGGT SGGGGSGGGGS L20- TCTGGTGGTG GTGGTTCTGG CGGTGGCGGT GGGGSGGGG- FGF21 TCTGGCGGCG GTGGTAGCGG TGGCGGCGGT FGF21(33- (V76) GATAGCAGCC CGCTGCTGCA GTTTGGCGGC 209; Q56E- CAGGTGCGTC AGCGTTATCT GTATACCGAT D74H-Q82E- GATGCGCAGG AAACCGAAGC GCATCTGGAA R105K- ATTCGTGAAG ATGGCACCGT GGGCGGTGCG K150R- GCGCATCAGA GCCCGGAAAG CCTGCTGGAA R154C- CTGAAAGCGC TGAAACCGGG CGTGATTCAG R159Q- ATTCTGGGCG TGAAAACCAG CCGTTTTCTG S195A) TGCCAGAAAC CGGATGGCGC GCTGTATGGC AGCCTGCATT TTGATCCGGA AGCGTGCAGC TTTCGTGAAC TGCTGCTGGA AGATGGCTAT AACGTGTATC AGAGCGAAGC GCATGGCCTG CCGCTGCATC TGCCGGGCAA CCGTAGCCCG CATTGCGATC CGGCACCGCA GGGTCCGGCG CGTTTTCTGC CGCTGCCGGG TCTGCCGCCG GCACTGCCGG AACCGCCGGG TATTCTGGCC CCGCAGCCGC CGGATGTTGG TAGCAGCGAT CCGCTGGCGA TGGTGGGTCC GAGCCAGGGT CGTAGCCCGA GCTATGCGAG CTAA 13 V220 HSEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7- GLP- SGDSSPLLQF GGQVRQRYLY TDDAQQTEAH 35; A8S)- 1(A8S)- LEIREDGTVG GAADQSPESL LQLKALKPGV GSG- L3- IQILGVKTSR FLCQRPDGAL YGSLHFDPEA FGF21  FGF21 CSFRELLLED GYNVYQSEAH GLPLHLPGNK (33-209) SPHRDPAPRG PARFLPLPGL PPALPEPPGI LAPQPPDVGS SDPLSMVGPS QGRSPSYAS 14 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7- GLP- TCTAGCTACC TGGAAGGCCA GGCTGCGAAA 35; A8S)- 1(A8S)- GAATTCATCG CGTGGCTGGT TAAAGGCGGT GSG- L3- TCTGGTGATA GCAGCCCGCT GCTGCAGTTT FGF21  FGF21 GGCGGCCAGG TGCGTCAGCG TTATCTGTAT (33-209) ACCGATGATG CGCAGCAGAC CGAAGCGCAT CTGGAAATTC GTGAAGATGG CACCGTGGGC GGTGCGGCGG ATCAGAGCCC GGAAAGCCTG CTGCAGCTGA AAGCGCTGAA ACCGGGCGTG ATTCAGATTC TGGGCGTGAA AACCAGCCGT TTTCTGTGCC AGCGTCCGGA TGGCGCGCTG TATGGCAGCC TGCATTTTGA TCCGGAAGCG TGCAGCTTTC GTGAACTGCT GCTGGAAGAT GGCTATAACG TGTATCAGAG CGAAGCGCAT GGCCTGCCGC TGCATCTGCC GGGCAACAAA AGCCCGCATC GTGATCCGGC ACCGCGTGGT CCGGCGCGTT TTCTGCCGCT GCCGGGTCTG CCGCCGGCAC TGCCGGAACC GCCGGGTATT CTGGCCCCGC AGCCGCCGGA TGTTGGTAGC AGCGATCCGC TGTCTATGGT GGGTCCGAGC CAGGGTCGTA GCCCGAGCTA TGCGAGCTAA 15 V219 HAEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7- GLP-1-  SGDSSPLLQF GGQVRQRYLY TDDAQQTEAH 35)-GSG- L3- LEIREDGTVG GAADQSPESL LQLKALKPGV FGF21  FGF21 IQILGVKTSR FLCQRPDGAL YGSLHFDPEA (33-209) CSFRELLLED GYNVYQSEAH GLPLHLPGNK SPHRDPAPRG PARFLPLPGL PPALPEPPGI LAPQPPDVGS SDPLSMVGPS QGRSPSYAS 16 CATGCGGAAG GCACTTTTAC TAGCGATGTT GLP-1(7- GLP-1-  TCTAGCTACC TGGAAGGCCA GGCTGCGAAA 35)-GSG- L3- GAATTCATCG CGTGGCTGGT TAAAGGCGGT FGF21  FGF21 TCTGGTGATA GCAGCCCGCT GCTGCAGTTT (33-209) GGCGGCCAGG TGCGTCAGCG TTATCTGTAT ACCGATGATG CGCAGCAGAC CGAAGCGCAT CTGGAAATTC GTGAAGATGG CACCGTGGGC GGTGCGGCGG ATCAGAGCCC GGAAAGCCTG CTGCAGCTGA AAGCGCTGAA ACCGGGCGTG ATTCAGATTC TGGGCGTGAA AACCAGCCGT TTTCTGTGCC AGCGTCCGGA TGGCGCGCTG TATGGCAGCC TGCATTTTGA TCCGGAAGCG TGCAGCTTTC GTGAACTGCT GCTGGAAGAT GGCTATAACG TGTATCAGAG CGAAGCGCAT GGCCTGCCGC TGCATCTGCC GGGCAACAAA AGCCCGCATC GTGATCCGGC ACCGCGTGGT CCGGCGCGTT TTCTGCCGCT GCCGGGTCTG CCGCCGGCAC TGCCGGAACC GCCGGGTATT CTGGCCCCGC AGCCGCCGGA TGTTGGTAGC AGCGATCCGC TGTCTATGGT GGGTCCGAGC CAGGGTCGTA GCCCGAGCTA TGCGAGCTAA 17 V295 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1- PSSGAPPPSG GGDSSPLLQF GGQVRQRYLY 39)-GGG- 39)-L3- TDDAQQTEAH LEIREDGTVG GAADQSPESL FGF21(33- FGF21 LQLKALKPGV IQILGVKTSR FLCQRPDGAL 209) YGSLHFDPEA CSFRELLLED GYNVYQSEAH GLPLHLPGNK SPHRDPAPRG PARFLPLPGL PPALPEPPGI LAPQPPDVGS SDPLSMVGPS QGRSPSYAS 18 CATGGTGAGG GTACGTTTAC TTCTGATCTG Exendin4(1- Ex(1- TCTAAACAGA TGGAAGAAGA AGCTGTTCGC 39)-GGG- 39)-L3- CTGTTCATTG AATGGCTGAA AAATGGTGGT FGF21 FGF21 CCGTCCTCCG GCGCTCCTCC GCCTTCTGGT (33-209) GGTGGCGACT CGAGCCCGCT GCTGCAGTTT GGCGGCCAGG TGCGTCAGCG TTATCTGTAT ACCGATGATG CGCAGCAGAC CGAAGCGCAT CTGGAAATTC GTGAAGATGG CACCGTGGGC GGTGCGGCGG ATCAGAGCCC GGAAAGCCTG CTGCAGCTGA AAGCGCTGAA ACCGGGCGTG ATTCAGATTC TGGGCGTGAA AACCAGCCGT TTTCTGTGCC AGCGTCCGGA TGGCGCGCTG TATGGCAGCC TGCATTTTGA TCCGGAAGCG TGCAGCTTTC GTGAACTGCT GCTGGAAGAT GGCTATAACG TGTATCAGAG CGAAGCGCAT GGCCTGCCGC TGCATCTGCC GGGCAACAAA AGCCCGCATC GTGATCCGGC ACCGCGTGGT CCGGCGCGTT TTCTGCCGCT GCCGGGTCTG CCGCCGGCAC TGCCGGAACC GCCGGGTATT CTGGCCCCGC AGCCGCCGGA TGTTGGTAGC AGCGATCCGC TGTCTATGGT GGGTCCGAGC CAGGGTCGTA GCCCGAGCTA TGCGAGCTAA 19 V224 HSEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7- GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD 35; A8S)- 1(A8S)- AQQTEAHLEI REDGTVGGAA DQSPESLLQL GSGGGGSGGG- L10- KALKPGVIQI LGVKTSRFLC QRPDGALYGS FGF21 FGF21 LHFDPEACSF RELLLEDGYN VYQSEAHGLP (33-209) LHLPGNKSPH RDPAPRGPAR FLPLPGLPPA LPEPPGILAP QPPDVGSSDP LSMVGPSQGR SPSYAS 20 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7- GLP- TCTAGCTACC TGGAAGGCCA GGCTGCGAAA 35; A8S)- 1(A8S)- GAATTCATCG CGTGGCTGGT TAAAGGCGGT GSGGGGSGGG- L10- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21  FGF21 TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG (33-209) GTGCGTCAGC GTTATCTGTA TACCGATGAT GCGCAGCAGA CCGAAGCGCA TCTGGAAATT CGTGAAGATG GCACCGTGGG CGGTGCGGCG GATCAGAGCC CGGAAAGCCT GCTGCAGCTG AAAGCGCTGA AACCGGGCGT GATTCAGATT CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGCGTCCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACAA AAGCCCGCAT CGTGATCCGG CACCGCGTGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGTCTATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 21 V223 HAEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7- GLP-1- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD 35)- L10- AQQTEAHLEI REDGTVGGAA DQSPESLLQL GSGGGGSGGG- FGF21 KALKPGVIQI LGVKTSRFLC QRPDGALYGS FGF21 LHFDPEACSF RELLLEDGYN VYQSEAHGLP (33-209) LHLPGNKSPH RDPAPRGPAR FLPLPGLPPA LPEPPGILAP QPPDVGSSDP LSMVGPSQGR SPSYAS 22 CATGCGGAAG GCACTTTTAC TAGCGATGTT GLP-1(7- GLP-1- TCTAGCTACC TGGAAGGCCA GGCTGCGAAA 35)- L10- GAATTCATCG CGTGGCTGGT TAAAGGCGGT GSGGGGSGGG- FGF21 TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21 TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG (33-209) GTGCGTCAGC GTTATCTGTA TACCGATGAT GCGCAGCAGA CCGAAGCGCA TCTGGAAATT CGTGAAGATG GCACCGTGGG CGGTGCGGCG GATCAGAGCC CGGAAAGCCT GCTGCAGCTG AAAGCGCTGA AACCGGGCGT GATTCAGATT CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGCGTCCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACAA AAGCCCGCAT CGTGATCCGG CACCGCGTGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGTCTATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 23 V234 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4 Ex(1- PSSGAPPPSG GGGSGGGDSS PLLQFGGQVR (1-39)- 39)-L8- QRYLYTDDAQ QTEAHLEIRE DGTVGGAADQ GGGGSGGG- FGF21 SPESLLQLKA LKPGVIQILG VKTSRFLCQR FGF21(33-209) PDGALYGSLH FDPEACSFRE LLLEDGYNVY QSEAHGLPLH LPGNKSPHRD PAPRGPARFL PLPGLPPALP EPPGILAPQP PDVGSSDPLS MVGPSQGRSP SYAS 24 CATGGTGAGG GTACGTTTAC TTCTGATCTG Exendin4 Ex(1- TCTAAACAGA TGGAAGAAGA AGCTGTTCGC (1-39)- 39)-L8- CTGTTCATTG AATGGCTGAA AAATGGTGGT GGGGSGGG- FGF21 CCGTCCTCCG GCGCTCCTCC GCCTTCTGGT FGF21(33-209) GGTGGTGGTT CTGGCGGTGG CGACTCGAGC CCGCTGCTGC AGTTTGGCGG CCAGGTGCGT CAGCGTTATC TGTATACCGA TGATGCGCAG CAGACCGAAG CGCATCTGGA AATTCGTGAA GATGGCACCG TGGGCGGTGC GGCGGATCAG AGCCCGGAAA GCCTGCTGCA GCTGAAAGCG CTGAAACCGG GCGTGATTCA GATTCTGGGC GTGAAAACCA GCCGTTTTCT GTGCCAGCGT CCGGATGGCG CGCTGTATGG CAGCCTGCAT TTTGATCCGG AAGCGTGCAG CTTTCGTGAA CTGCTGCTGG AAGATGGCTA TAACGTGTAT CAGAGCGAAG CGCATGGCCT GCCGCTGCAT CTGCCGGGCA ACAAAAGCCC GCATCGTGAT CCGGCACCGC GTGGTCCGGC GCGTTTTCTG CCGCTGCCGG GTCTGCCGCC GGCACTGCCG GAACCGCCGG GTATTCTGGC CCCGCAGCCG CCGGATGTTG GTAGCAGCGA TCCGCTGTCT ATGGTGGGTC CGAGCCAGGG TCGTAGCCCG AGCTATGCGA GCTAA 25 V193 HSEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7- GLP- SGGGGSGGGG SGGGGSADKT HTCPPCPAPE 35; A8S)- 1(A8S)- AAGGPSVFLF PPKPKDTLMI SRTPEVTCVV S(GGGGS)₃A- L17-Fc- VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE Fc-(GGGGS)₃- L15- EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV FGF21(33- FGF21 SNKALPAPIE KTISKAKGQP REPQVYTLPP 209; Q55C- (V103) SREEMTKNQV SLTCLVKGFY PSDIAVEWES R105K- NGQPENNYKT TPPVLDSDGS FFLYSKLTVD G148C- KSRWQQGNVF SCSVMHEALH NHYTQKSLSL K150R- SPGKGGGGSG GGGSGGGGSD SSPLLQFGGQ P158S- VRQRYLYTDD ACQTEAHLEI REDGTVGGAA 5195A- DQSPESLLQL KALKPGVIQI LGVKTSRFLC P199G- QKPDGALYGS LHFDPEACSF RELLLEDGYN G202A) VYQSEAHGLP LHLPCNRSPH RDPASRGPAR FLPLPGLPPA LPEPPGILAP QPPDVGSSDP LAMVGGSQAR SPSYAS 26 V194 HSEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7- GLP- SGGGGSGGGG SGGGGSADKT HTCPPCPAPE 35; A8S)- 1(A8S)- AAGGPSVFLF PPKPKDTLMI SRTPEVTCVV S(GGGGS)₃A- L17-Fc- VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE Fc-(GGGGS)₃- L15- EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV FGF21(33- FGF21 SNKALPAPIE KTISKAKGQP REPQVYTLPP 209; Q55C- (V194) SREEMTKNQV SLTCLVKGFY PSDIAVEWES R105K- NGQPENNYKT TPPVLDSDGS FFLYSKLTVD G148C- KSRWQQGNVF SCSVMHEALH NHYTQKSLSL K150R- SPGKGGGGSG GGGSGGGGSD SSPLLQFGGQ P158S- VRQRYLYTDD ACQTEAHLEI REDGTVGGAA S195A- DQSPESLLQL KALKPGVIQI LGVKTSRFLC G202A- QKPDGALYGS LHFDPEACSF RELLLEDGYN A208E) VYQSEAHGLP LHLPCNRSPH RDPASRGPAR FLPLPGLPPA LPEPPGILAP QPPDVGSSDP LAMVGPSQAR SPSYES 27 V195 HSEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7- GLP- SGGGGSGGGG SGGGGSADKT HTCPPCPAPE 35; A8S)- 1(A8S)- AAGGPSVFLF PPKPKDTLMI SRTPEVTCVV S(GGGGS)₃A- L17-Fc- VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE Fc-(GGGGS)₃- L15- EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV FGF21(33- FGF21 SNKALPAPIE KTISKAKGQP REPQVYTLPP 209; Q55C- (V195) SREEMTKNQV SLTCLVKGFY PSDIAVEWES R105K- NGQPENNYKT TPPVLDSDGS FFLYSKLTVD G148C- KSRWQQGNVF SCSVMHEALH NHYTQKSLSL P158S- SPGKGGGGSG GGGSGGGGSD SSPLLQFGGQ K150R- VRQRYLYTDD ACQTEAHLEI REDGTVGGAA S195A- DQSPESLLQL KALKPGVIQI LGVKTSRFLC G202A- QKPDGALYGS LHFDPEACSF RELLLEDGYN A208D) VYQSEAHGLP LHLPCNRSPH RDPASRGPAR FLPLPGLPPA LPEPPGILAP QPPDVGSSDP LAMVGPSQAR SPSYDS 28 V296 HGEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7- GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD 35; A8G)- 1(A8G)- AQETEAHLEI REDGTVGGAA HQSPESLLEL GSGGGGSGGG- L10- KALKPGVIQI LGVKTSRFLC QKPDGALYGS FGF21(33- FGF21 LHFDPEACSF RELLLEDGYN VYQSEAHGLP 209; Q56E- (V76)- LHLPGNRSPH CDPAPQGPAR FLPLPGLPPA D74H-Q82E- 40KPEGb LPEPPGILAP QPPDVGSSDP LAMVGPSQGR R105K- SPSYAS K150R- R154C-40 kDa branched PEG-R159Q- S195A) 29 CATGGTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7- GLP- TCTAGCTACC TGGAAGGCCA GGCTGCGAAA 35; A8G)- 1(A8G)- GAATTCATCG CGTGGCTGGT TAAAGGCGGT GSGGGGSGGG- L10- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAT FGF21(33- FGF21 AGCAGCCCGC TGCTGCAGTT TGGCGGCCAG 209; Q56E- (V76) GTGCGTCAGC GTTATCTGTA TACCGATGAT D74H-Q82E- GCGCAGGAAA CCGAAGCGCA TCTGGAAATT R105K- CGTGAAGATG GCACCGTGGG CGGTGCGGCG K150R- CATCAGAGCC CGGAAAGCCT GCTGGAACTG R154C- AAAGCGCTGA AACCGGGCGT GATTCAGATT R159Q- CTGGGCGTGA AAACCAGCCG TTTTCTGTGC S195A) CAGAAACCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACCG TAGCCCGCAT TGCGATCCGG CACCGCAGGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGGCGATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 34 V294 HSEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7- GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD 35; A8S)- 1(A8S)- AQQTEAHLEI REDGTVGGAA DQSPESLLQL GSGGGGSGGG- L10- KALKPGVIQI LGVKTSRFLC QRPDGALYGS FGF21(33- FGF21- LHFDPEACSF RELLLEDGYN VYQSEAHGLP 209;  154Pcl- LHLPGNKSPH UDPAPRGPAR FLPLPGLPPA R154Pcl-40 40KPEGb LPEPPGILAP QPPDVGSSDP LSMVGPSQGR kDa SPSYAS branched PEG) 35 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7- GLP- TCTAGCTACC TGGAAGGCCA GGCTGCGAAA 35; A8S)- 1(A8S)- GAATTCATCG CGTGGCTGGT TAAAGGCGGT GSGGGGSGGG- L10- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21(33- FGF21- TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG 209;  154TAG GTGCGTCAGC GTTATCTGTA TACCGATGAT R154TAG) GCGCAGCAGA CCGAAGCGCA TCTGGAAATT CGTGAAGATG GCACCGTGGG CGGTGCGGCG GATCAGAGCC CGGAAAGCCT GCTGCAGCTG AAAGCGCTGA AACCGGGCGT GATTCAGATT CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGCGTCCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACAA AAGCCCGCAT TAGGATCCGG CACCGCGTGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGTCTATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 38 V298 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4 Ex(1- PSSGAPPPSG GGGSGGGDSS PLLQFGGQVR (1-39)- 39)-L8- QRYLYTDDAQ QTEAHLEIRE DGTVGGAADQ GGGGSGGG- FGF21 SPESLLQLKA LKPGVIQILG VKTSRFLCQR FGF21(33- (154Pcl)- PDGALYGSLH FDPEACSFRE LLLEDGYNVY 209;  40KPEGb QSEAHGLPLH LPGNKSPHUD PAPRGPARFL R154TAG)-40 PLPGLPPALP EPPGILAPQP PDVGSSDPLS kDa MVGPSQGRSP SYAS branched PEG 39 CATGGTGAGG GTACGTTTAC TTCTGATCTG Exendin4 Ex(1- TCTAAACAGA TGGAAGAAGA AGCTGTTCGC (1-39)- 39)-L8- CTGTTCATTG AATGGCTGAA AAATGGTGGT GGGGSGGG- FGF21 CCGTCCTCCG GCGCTCCTCC GCCTTCTGGT FGF21(33- (154TAG) GGTGGTGGTT CTGGCGGTGG CGACTCGAGC 209;  CCGCTGCTGC AGTTTGGCGG CCAGGTGCGT R154TAG) CAGCGTTATC TGTATACCGA TGATGCGCAG CAGACCGAAG CGCATCTGGA AATTCGTGAA GATGGCACCG TGGGCGGTGC GGCGGATCAG AGCCCGGAAA GCCTGCTGCA GCTGAAAGCG CTGAAACCGG GCGTGATTCA GATTCTGGGC GTGAAAACCA GCCGTTTTCT GTGCCAGCGT CCGGATGGCG CGCTGTATGG CAGCCTGCAT TTTGATCCGG AAGCGTGCAG CTTTCGTGAA CTGCTGCTGG AAGATGGCTA TAACGTGTAT CAGAGCGAAG CGCATGGCCT GCCGCTGCAT CTGCCGGGCA ACAAAAGCCC GCATTAGGAT CCGGCACCGC GTGGTCCGGC GCGTTTTCTG CCGCTGCCGG GTCTGCCGCC GGCACTGCCG GAACCGCCGG GTATTCTGGC CCCGCAGCCG CCGGATGTTG GTAGCAGCGA TCCGCTGTCT ATGGTGGGTC CGAGCCAGGG TCGTAGCCCG AGCTATGCGA GCTAA 40 V235 HSEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7-35;  GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD A8S)- 1(A8S)- AQQTEAHLEI REDGTVGGAA DQSPESLLQL GSGGGGSGGG- L8- KALKPGVIQI LGVKTSRFLC QRPDGALYGS FGF21(33- FGF21 LHFDPEACSF RELLLEDGYN VYQSEAHGLP 209; R154C- (154C)- LHLPGNKSPH CDPAPRGPAR FLPLPGLPPA 40 kDa 40KPEGb LPEPPGILAP QPPDVGSSDP LSMVGPSQGR branched SPSYAS PEG) 41 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7-35;  GLP- TCTAGCTACC TGGAAGGCCA GGCTGCGAAA A8S)- 1(A8S)- GAATTCATCG CGTGGCTGGT TAAAGGCGGT GSGGGGSGGG- L8- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21(33- FGF21(1 TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG 209; R154C) 54C) GTGCGTCAGC GTTATCTGTA TACCGATGAT GCGCAGCAGA CCGAAGCGCA TCTGGAAATT CGTGAAGATG GCACCGTGGG CGGTGCGGCG GATCAGAGCC CGGAAAGCCT GCTGCAGCTG AAAGCGCTGA AACCGGGCGT GATTCAGATT CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGCGTCCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACAA AAGCCCGCAT TGCGATCCGG CACCGCGTGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGTCTATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 42 V239 HAEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7- GLP-1- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD 35)- L8- AQQTEAHLEI REDGTVGGAA DQSPESLLQL GSGGGGSGGG- FGF21 KALKPGVIQI LGVKTSRFLC QRPDGALYGS FGF21(33- (154C)- LHFDPEACSF RELLLEDGYN VYQSEAHGLP 209; R154C- 40KPEGb LHLPGNKSPH CDPAPRGPAR FLPLPGLPPA 40 kDa LPEPPGILAP QPPDVGSSDP LSMVGPSQGR branched SPSYAS PEG) 43 CATGCGGAAG GCACTTTTAC TAGCGATGTT GLP-1(7- GLP-1- TCTAGCTACC TGGAAGGCCA GGCTGCGAAA 35)- L8- GAATTCATCG CGTGGCTGGT TAAAGGCGGT GSGGGGSGGG- FGF21 TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21(33- (154C) TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG 209; R154C) GTGCGTCAGC GTTATCTGTA TACCGATGAT GCGCAGCAGA CCGAAGCGCA TCTGGAAATT CGTGAAGATG GCACCGTGGG CGGTGCGGCG GATCAGAGCC CGGAAAGCCT GCTGCAGCTG AAAGCGCTGA AACCGGGCGT GATTCAGATT CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGCGTCCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACAA AAGCCCGCAT TGCGATCCGG CACCGCGTGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGTCTATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 44 V299 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4 Ex(1- PSSGAPPPSG GGGSGGGDSS PLLQFGGQVR (1-39)- 39)-L8- QRYLYTDDAQ QTEAHLEIRE DGTVGGAADQ GGGGSGGG- FGF21 SPESLLQLKA LKPGVIQILG VKTSRFLCQR FGF21(33- (154C)- PDGALYGSLH FDPEACSFRE LLLEDGYNVY 209; R154C- 40KPEGb QSEAHGLPLH LPGNKSPHCD PAPRGPARFL 40 kDa PLPGLPPALP EPPGILAPQP PDVGSSDPLS branched MVGPSQGRSP SYAS PEG) 45 CATGGTGAGG GTACGTTTAC TTCTGATCTG Exendin4 Ex(1- TCTAAACAGA TGGAAGAAGA AGCTGTTCGC (1-39)- 39)-L8- CTGTTCATTG AATGGCTGAA AAATGGTGGT GGGGSGGG- FGF21 CCGTCCTCCG GCGCTCCTCC GCCTTCTGGT FGF21(33- (154C) GGTGGTGGTT CTGGCGGTGG CGACTCGAGC 209; R154C) CCGCTGCTGC AGTTTGGCGG CCAGGTGCGT CAGCGTTATC TGTATACCGA TGATGCGCAG CAGACCGAAG CGCATCTGGA AATTCGTGAA GATGGCACCG TGGGCGGTGC GGCGGATCAG AGCCCGGAAA GCCTGCTGCA GCTGAAAGCG CTGAAACCGG GCGTGATTCA GATTCTGGGC GTGAAAACCA GCCGTTTTCT GTGCCAGCGT CCGGATGGCG CGCTGTATGG CAGCCTGCAT TTTGATCCGG AAGCGTGCAG CTTTCGTGAA CTGCTGCTGG AAGATGGCTA TAACGTGTAT CAGAGCGAAG CGCATGGCCT GCCGCTGCAT CTGCCGGGCA ACAAAAGCCC GCATTGCGAT CCGGCACCGC GTGGTCCGGC GCGTTTTCTG CCGCTGCCGG GTCTGCCGCC GGCACTGCCG GAACCGCCGG GTATTCTGGC CCCGCAGCCG CCGGATGTTG GTAGCAGCGA TCCGCTGTCT ATGGTGGGTC CGAGCCAGGG TCGTAGCCCG AGCTATGCGA GCTAA 46 V271 HAPGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7-35;  GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD E9P)- 1(E9P)- AQQTEAHLEI REDGTVGGAA DQSPESLLQL GSGGGGSGGG- L10- KALKPGVIQI LGVKTSRFLC QRPDGALYGS FGF21(33- FGF21 LHFDPEACSF RELLLEDGYN VYQSEAHGLP 209; R154C- (154C)- LHLPGNKSPH CDPAPRGPAR FLPLPGLPPA 40 kDa 40KPEGb LPEPPGILAP QPPDVGSSDP LSMVGPSQGR branched SPSYAS PEG) 47 CATGCGCCGG GCACTTTTAC TAGCGATGTT GLP-1(7-35;  GLP- TCTAGCTACC TGGAAGGCCA GGCTGCGAAA E9P)- 1(E9P)- GAATTCATCG CGTGGCTGGT TAAAGGCGGT GSGGGGSGGG- L10- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21(33- FGF21 TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG 209; R154C) (154C) GTGCGTCAGC GTTATCTGTA TACCGATGAT GCGCAGCAGA CCGAAGCGCA TCTGGAAATT CGTGAAGATG GCACCGTGGG CGGTGCGGCG GATCAGAGCC CGGAAAGCCT GCTGCAGCTG AAAGCGCTGA AACCGGGCGT GATTCAGATT CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGCGTCCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACAA AAGCCCGCAT TGCGATCCGG CACCGCGTGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGTCTATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 48 V251 GHAEGTFTSD VSSYLEGQAA KEFIAWLVKG GLP-1(6-35;  G-GLP- GSGGGGSGGG DSSPLLQFGG QVRQRYLYTD 6G)- 1-L10- DAQQTEAHLE IREDGTVGGA ADQSPESLLQ GSGGGGSGGG- FGF21 LKALKPGVIQ ILGVKTSRFL CQRPDGALYG FGF21(33- (154C)- SLHFDPEACS FRELLLEDGY NVYQSEAHGL 209; R154C- 40KPEGb PLHLPGNKSP HCDPAPRGPA RFLPLPGLPP 40 kDa ALPEPPGILA PQPPDVGSSD PLSMVGPSQG branched RSPSYAS PEG) 49 GGTCATGCGG AAGGCACTTT TACTAGCGAT GLP-1(6-35;  G-GLP- GTTTCTAGCT ACCTGGAAGG CCAGGCTGCG 6G)- 1-L10- AAAGAATTCA TCGCGTGGCT GGTTAAAGGC GSGGGGSGGG- FGF21 GGTTCTGGTG GTGGTGGTTC TGGCGGTGGC FGF21(33- (154C) GACTCGAGCC CGCTGCTGCA GTTTGGCGGC 209) CAGGTGCGTC AGCGTTATCT GTATACCGAT GATGCGCAGC AGACCGAAGC GCATCTGGAA ATTCGTGAAG ATGGCACCGT GGGCGGTGCG GCGGATCAGA GCCCGGAAAG CCTGCTGCAG CTGAAAGCGC TGAAACCGGG CGTGATTCAG ATTCTGGGCG TGAAAACCAG CCGTTTTCTG TGCCAGCGTC CGGATGGCGC GCTGTATGGC AGCCTGCATT TTGATCCGGA AGCGTGCAGC TTTCGTGAAC TGCTGCTGGA AGATGGCTAT AACGTGTATC AGAGCGAAGC GCATGGCCTG CCGCTGCATC TGCCGGGCAA CAAAAGCCCG CATTGCGATC CGGCACCGCG TGGTCCGGCG CGTTTTCTGC CGCTGCCGGG TCTGCCGCCG GCACTGCCGG AACCGCCGGG TATTCTGGCC CCGCAGCCGC CGGATGTTGG TAGCAGCGAT CCGCTGTCTA TGGTGGGTCC GAGCCAGGGT CGTAGCCCGA GCTATGCGAG CTAA 50 V265 HSEGTFTADA SSYLEGQAAK EFIAWLVKGG GLP-1(7-35;  GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD A8S-S14A- 1(A8S;  AQQTEAHLEI REDGTVGGAA DQSPESLLQL V16A)- 14/16)- KALKPGVIQI LGVKTSRFLC QRPDGALYGS GSGGGGSGGG- L10- LHFDPEACSF RELLLEDGYN VYQSEAHGLP FGF21(33- FGF21 LHLPGNKSPH CDPAPRGPAR FLPLPGLPPA 209; R154C- (154C)- LPEPPGILAP QPPDVGSSDP LSMVGPSQGR 40 kDa 40KPEGb SPSYAS branched PEG) 51 CATTCTGAAG GCACTTTTAC TGCTGATGCT GLP-1(7-35;  GLP- TCTAGCTACC TGGAAGGCCA GGCTGCGAAA A8S-S14A- 1(A8S;  GAATTCATCG CGTGGCTGGT TAAAGGCGGT V16A)- 14/16)- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC GSGGGGSGGG- L10- TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG FGF21(33- FGF21 GTGCGTCAGC GTTATCTGTA TACCGATGAT 209; R154C) (154C) GCGCAGCAGA CCGAAGCGCA TCTGGAAATT CGTGAAGATG GCACCGTGGG CGGTGCGGCG GATCAGAGCC CGGAAAGCCT GCTGCAGCTG AAAGCGCTGA AACCGGGCGT GATTCAGATT CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGCGTCCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACAA AAGCCCGCAT TGCGATCCGG CACCGCGTGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGTCTATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 52 V270 HSEGTFTSDA AAYLEGQAAK EFIAWLVKGG GLP-1(7-35;  GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD A8S-V16A- 1(A8S;  AQQTEAHLEI REDGTVGGAA DQSPESLLQL S17A-S18A)- 16/17/ KALKPGVIQI LGVKTSRFLC QRPDGALYGS GSGGGGSGGG- 18)- LHFDPEACSF RELLLEDGYN VYQSEAHGLP FGF21(33- L10- LHLPGNKSPH CDPAPRGPAR FLPLPGLPPA 209; R154C- FGF21 LPEPPGILAP QPPDVGSSDP LSMVGPSQGR 40 kDa (154C)- SPSYAS branched 40KPEGb PEG) 53 CATTCTGAAG GCACTTTTAC TAGCGATGCT GLP-1(7-35;  GLP- GCTGCTTACC TGGAAGGCCA GGCTGCGAAA A8S-V16A- 1(A8S)- GAATTCATCG CGTGGCTGGT TAAAGGCGGT S17A-S18A)- 16/17/ TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC GSGGGGSGGG- 18)- TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG FGF21(33- L10- GTGCGTCAGC GTTATCTGTA TACCGATGAT 209; R154C) (1FGF21 GCGCAGCAGA CCGAAGCGCA TCTGGAAATT 54C) CGTGAAGATG GCACCGTGGG CGGTGCGGCG GATCAGAGCC CGGAAAGCCT GCTGCAGCTG AAAGCGCTGA AACCGGGCGT GATTCAGATT CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGCGTCCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACAA AAGCCCGCAT TGCGATCCGG CACCGCGTGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGTCTATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 54 V266 HSEGTFTSDV SSYAEGAAAK EFIAWLVKGG GLP-1(7-35;  GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD A8S-L20A- 1(A8S;  AQQTEAHLEI REDGTVGGAA DQSPESLLQL Q23A)- 20/23)- KALKPGVIQI LGVKTSRFLC QRPDGALYGS GSGGGGSGGG- L10- LHFDPEACSF RELLLEDGYN VYQSEAHGLP FGF21(33- FGF21 LHLPGNKSPH CDPAPRGPAR FLPLPGLPPA 209; R154C- (154C)- LPEPPGILAP QPPDVGSSDP LSMVGPSQGR 40 kDa 40KPEGb SPSYAS branched PEG) 55 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7-35;  GLP- TCTAGCTACG CTGAAGGCGC TGCTGCGAAA A8S-L20A- 1(A8S;  GAATTCATCG CGTGGCTGGT TAAAGGCGGT Q23A)- 20/23)- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC GSGGGGSGGG- L10- TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG FGF21(33- FGF21 GTGCGTCAGC GTTATCTGTA TACCGATGAT 209; R154C) (154C) GCGCAGCAGA CCGAAGCGCA TCTGGAAATT CGTGAAGATG GCACCGTGGG CGGTGCGGCG GATCAGAGCC CGGAAAGCCT GCTGCAGCTG AAAGCGCTGA AACCGGGCGT GATTCAGATT CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGCGTCCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACAA AAGCCCGCAT TGCGATCCGG CACCGCGTGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGTCTATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 58 V300 HSEGTFTSDS SPLLQFGGQV RQRYLYTDDA GLP-1(7-14;  GLP- QQTEAHLEIR EDGTVGGAAD QSPESLLQLK A8S)- 1(A8S;  ALKPGVIQIL GVKTSRFLCQ RPDGALYGSL FGF21(33- 7-14)- HFDPEACSFR ELLLEDGYNV YQSEAHGLPL 209; R154C- L0- HLPGNKSPHC DPAPRGPARF LPLPGLPPAL 40 kDa FGF21 PEPPGILAPQ PPDVGSSDPL SMVGPSQGRS branched (154C)- PSYAS PEG) 40KPEGb 59 CATTCTGAAG GCACTTTTAC TAGCGATAGC GLP-1(7-14;  GLP- AGCCCGCTGC TGCAGTTTGG CGGCCAGGTG A8S)- 1(A8S;  CGTCAGCGTT ATCTGTATAC CGATGATGCG FGF21(33- 7-14)- CAGCAGACCG AAGCGCATCT GGAAATTCGT 209; R154C) L0- GAAGATGGCA CCGTGGGCGG TGCGGCGGAT FGF21 CAGAGCCCGG AAAGCCTGCT GCAGCTGAAA (154C) GCGCTGAAAC CGGGCGTGAT TCAGATTCTG GGCGTGAAAA CCAGCCGTTT TCTGTGCCAG CGTCCGGATG GCGCGCTGTA TGGCAGCCTG CATTTTGATC CGGAAGCGTG CAGCTTTCGT GAACTGCTGC TGGAAGATGG CTATAACGTG TATCAGAGCG AAGCGCATGG CCTGCCGCTG CATCTGCCGG GCAACAAAAG CCCGCATTGC GATCCGGCAC CGCGTGGTCC GGCGCGTTTT CTGCCGCTGC CGGGTCTGCC GCCGGCACTG CCGGAACCGC CGGGTATTCT GGCCCCGCAG CCGCCGGATG TTGGTAGCAG CGATCCGCTG TCTATGGTGG GTCCGAGCCA GGGTCGTAGC CCGAGCTATG CGAGCTAA 60 V262 HSEGTFTSDV SSGGGGSGGG DSSPLLQFGG GLP-1(7-18;  GLP- QVRQRYLYTD DAQQTEAHLE IREDGTVGGA A8S)- 1(A8S;  ADQSPESLLQ LKALKPGVIQ ILGVKTSRFL GGGGSGGG- 7-18)- CQRPDGALYG SLHFDPEACS FRELLLEDGY FGF21(33- L8- NVYQSEAHGL PLHLPGNKSP HCDPAPRGPA 209; R154C- FGF21 RFLPLPGLPP ALPEPPGILA PQPPDVGSSD 40 kDa (154C)- PLSMVGPSQG RSPSYAS branched 40KPEGb PEG) 61 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7-18;  GLP- TCTTCTGGTG GTGGTGGTTC TGGCGGTGGC A8S)- 1(A8S;  GACTCGAGCC CGCTGCTGCA GTTTGGCGGC GGGGSGGG- 7-18)- CAGGTGCGTC AGCGTTATCT GTATACCGAT FGF21(33- L8- GATGCGCAGC AGACCGAAGC GCATCTGGAA 209; R154C) FGF21 ATTCGTGAAG ATGGCACCGT GGGCGGTGCG (154C) GCGGATCAGA GCCCGGAAAG CCTGCTGCAG CTGAAAGCGC TGAAACCGGG CGTGATTCAG ATTCTGGGCG TGAAAACCAG CCGTTTTCTG TGCCAGCGTC CGGATGGCGC GCTGTATGGC AGCCTGCATT TTGATCCGGA AGCGTGCAGC TTTCGTGAAC TGCTGCTGGA AGATGGCTAT AACGTGTATC AGAGCGAAGC GCATGGCCTG CCGCTGCATC TGCCGGGCAA CAAAAGCCCG CATTGCGATC CGGCACCGCG TGGTCCGGCG CGTTTTCTGC CGCTGCCGGG TCTGCCGCCG GCACTGCCGG AACCGCCGGG TATTCTGGCC CCGCAGCCGC CGGATGTTGG TAGCAGCGAT CCGCTGTCTA TGGTGGGTCC GAGCCAGGGT CGTAGCCCGA GCTATGCGAG CTAA 62 V261 HSEGTFTSDV SSYLESGGGG SGGGDSSPLL GLP-1(7-21;  GLP- QFGGQVRQRY LYTDDAQQTE AHLEIREDGT A8S)- 1(A8S;  VGGAADQSPE SLLQLKALKP GVIQILGVKT SGGGGSGGG- 7-21)- SRFLCQRPDG ALYGSLHFDP EACSFRELLL FGF21(33- L9- EDGYNVYQSE AHGLPLHLPG NKSPHCDPAP 209; R154C- FGF21 RGPARFLPLP GLPPALPEPP GILAPQPPDV 40 kDa (154C)- GSSDPLSMVG PSQGRSPSYA S branched 40KPEGb PEG) 63 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7-21;  GLP- TCTAGCTACC TGGAATCTGG TGGTGGTGGT A8S)- 1(A8S;  TCTGGCGGTG GCGACTCGAG CCCGCTGCTG SGGGGSGGG- 7-21)- CAGTTTGGCG GCCAGGTGCG TCAGCGTTAT FGF21(33- L9- CTGTATACCG ATGATGCGCA GCAGACCGAA 209; R154C) FGF21 GCGCATCTGG AAATTCGTGA AGATGGCACC (154C) GTGGGCGGTG CGGCGGATCA GAGCCCGGAA AGCCTGCTGC AGCTGAAAGC GCTGAAACCG GGCGTGATTC AGATTCTGGG CGTGAAAACC AGCCGTTTTC TGTGCCAGCG TCCGGATGGC GCGCTGTATG GCAGCCTGCA TTTTGATCCG GAAGCGTGCA GCTTTCGTGA ACTGCTGCTG GAAGATGGCT ATAACGTGTA TCAGAGCGAA GCGCATGGCC TGCCGCTGCA TCTGCCGGGC AACAAAAGCC CGCATTGCGA TCCGGCACCG CGTGGTCCGG CGCGTTTTCT GCCGCTGCCG GGTCTGCCGC CGGCACTGCC GGAACCGCCG GGTATTCTGG CCCCGCAGCC GCCGGATGTT GGTAGCAGCG ATCCGCTGTC TATGGTGGGT CCGAGCCAGG GTCGTAGCCC GAGCTATGCG AGCTAA 66 V260 HSEGTFTSDV SSYLEGQAAK EFISGGGGSG GLP-1(7-29;  GLP- GGDSSPLLQF GGQVRQRYLY TDDAQQTEAH A8S)- 1(A8S;  LEIREDGTVG GAADQSPESL LQLKALKPGV SGGGGSGGG- 7-29)- IQILGVKTSR FLCQRPDGAL YGSLHFDPEA FGF21(33- L9- CSFRELLLED GYNVYQSEAH GLPLHLPGNK 209; R154C- FGF21 SPHCDPAPRG PARFLPLPGL PPALPEPPGI 40 kDa (154C)- LAPQPPDVGS SDPLSMVGPS QGRSPSYAS branched 40KPEGb PEG) 67 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7-29;  GLP- TCTAGCTACC TGGAAGGCCA GGCTGCGAAA A8S)- 1(A8S;  GAATTCATCT CTGGTGGTGG TGGTTCTGGC SGGGGSGGG- 7-29)- GGTGGCGACT CGAGCCCGCT GCTGCAGTTT FGF21(33- L9- GGCGGCCAGG TGCGTCAGCG TTATCTGTAT 209) FGF21 ACCGATGATG CGCAGCAGAC CGAAGCGCAT (154C) CTGGAAATTC GTGAAGATGG CACCGTGGGC GGTGCGGCGG ATCAGAGCCC GGAAAGCCTG CTGCAGCTGA AAGCGCTGAA ACCGGGCGTG ATTCAGATTC TGGGCGTGAA AACCAGCCGT TTTCTGTGCC AGCGTCCGGA TGGCGCGCTG TATGGCAGCC TGCATTTTGA TCCGGAAGCG TGCAGCTTTC GTGAACTGCT GCTGGAAGAT GGCTATAACG TGTATCAGAG CGAAGCGCAT GGCCTGCCGC TGCATCTGCC GGGCAACAAA AGCCCGCATT GCGATCCGGC ACCGCGTGGT CCGGCGCGTT TTCTGCCGCT GCCGGGTCTG CCGCCGGCAC TGCCGGAACC GCCGGGTATT CTGGCCCCGC AGCCGCCGGA TGTTGGTAGC AGCGATCCGC TGTCTATGGT GGGTCCGAGC CAGGGTCGTA GCCCGAGCTA TGCGAGCTAA 68 V259 HSEGTFTSDV SSYLEGQAAK EFIAWLSGGG GLP-1(7-32;  GLP- GSGGGDSSPL LQFGGQVRQR YLYTDDAQQT A8S)- 1(A8S;  EAHLEIREDG TVGGAADQSP ESLLQLKALK SGGGGSGGG- 7-32)- PGVIQILGVK TSRFLCQRPD GALYGSLHFD FGF21(33- L9- PEACSFRELL LEDGYNVYQS EAHGLPLHLP 209; R154C- FGF21 GNKSPHCDPA PRGPARFLPL PGLPPALPEP 40 kDa (154C)- PGILAPQPPD VGSSDPLSMV GPSQGRSPSY branched 40KPEGb AS PEG) 69 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7-32;  GLP- TCTAGCTACC TGGAAGGCCA GGCTGCGAAA A8S)- 1(A8S;  GAATTCATCG CGTGGCTGTC TGGTGGTGGT SGGGGSGGG- 7-32)- GGTTCTGGCG GTGGCGACTC GAGCCCGCTG FGF21(33- L9- CTGCAGTTTG GCGGCCAGGT GCGTCAGCGT 209; R154C) FGF21 TATCTGTATA CCGATGATGC GCAGCAGACC (154C) GAAGCGCATC TGGAAATTCG TGAAGATGGC ACCGTGGGCG GTGCGGCGGA TCAGAGCCCG GAAAGCCTGC TGCAGCTGAA AGCGCTGAAA CCGGGCGTGA TTCAGATTCT GGGCGTGAAA ACCAGCCGTT TTCTGTGCCA GCGTCCGGAT GGCGCGCTGT ATGGCAGCCT GCATTTTGAT CCGGAAGCGT GCAGCTTTCG TGAACTGCTG CTGGAAGATG GCTATAACGT GTATCAGAGC GAAGCGCATG GCCTGCCGCT GCATCTGCCG GGCAACAAAA GCCCGCATTG CGATCCGGCA CCGCGTGGTC CGGCGCGTTT TCTGCCGCTG CCGGGTCTGC CGCCGGCACT GCCGGAACCG CCGGGTATTC TGGCCCCGCA GCCGCCGGAT GTTGGTAGCA GCGATCCGCT GTCTATGGTG GGTCCGAGCC AGGGTCGTAG CCCGAGCTAT GCGAGCTAA 70 V263 HSEGTFTSDV CSYLEGQAAK EFIAWLVKGG GLP-1(7-35;  GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD A8S-S17C- 1(A8S- AQQTEAHLEI REDGTVGGAA DQSPESLLQL 40 kDa S17C)- KALKPGVIQI LGVKTSRFLC QRPDGALYGS branched 40KPEGb- LHFDPEACSF RELLLEDGYN VYQSEAHGLP PEG)- L10- LHLPGNKSPH RDPAPRGPAR FLPLPGLPPA GSGGGGSGGG- FGF21 LPEPPGILAP QPPDVGSSDP LSMVGPSQGR FGF21(33-209) SPSYAS 71 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7-35;  GLP- TGTAGCTACC TGGAAGGCCA GGCTGCGAAA A8S-S17C)- 1(A8S- GAATTCATCG CGTGGCTGGT TAAAGGCGGT GSGGGGSGGG- S17C)- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21(33- L10- TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG 209) FGF21 GTGCGTCAGC GTTATCTGTA TACCGATGAT GCGCAGCAGA CCGAAGCGCA TCTGGAAATT CGTGAAGATG GCACCGTGGG CGGTGCGGCG GATCAGAGCC CGGAAAGCCT GCTGCAGCTG AAAGCGCTGA AACCGGGCGT GATTCAGATT CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGCGTCCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACAA AAGCCCGCAT CGTGATCCGG CACCGCGTGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGTCTATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 72 V269 HSEGTFTSDV SSYLEGCAAK EFIAWLVKGG GLP-1(7-35;  GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD A8S-Q23C-40 1(A8S- AQQTEAHLEI REDGTVGGAA DQSPESLLQL kDa Q23C)- KALKPGVIQI LGVKTSRFLC QRPDGALYGS branched 40KPEGb- LHFDPEACSF RELLLEDGYN VYQSEAHGLP PEG)- L10- LHLPGNKSPH RDPAPRGPAR FLPLPGLPPA GSGGGGSGGG- FGF21 LPEPPGILAP QPPDVGSSDP LSMVGPSQGR FGF21(33- SPSYAS 209) 73 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7-35;  GLP- TCTAGCTACC TGGAAGGCTG TGCTGCGAAA A8S-Q23C)- 1(A8S- GAATTCATCG CGTGGCTGGT TAAAGGCGGT GSGGGGSGGG- Q23C)- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21(33- L10- TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG 209) FGF21 GTGCGTCAGC GTTATCTGTA TACCGATGAT GCGCAGCAGA CCGAAGCGCA TCTGGAAATT CGTGAAGATG GCACCGTGGG CGGTGCGGCG GATCAGAGCC CGGAAAGCCT GCTGCAGCTG AAAGCGCTGA AACCGGGCGT GATTCAGATT CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGCGTCCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACAA AAGCCCGCAT CGTGATCCGG CACCGCGTGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGTCTATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 74 V243 HSEGTFTSDV SSYLEGQAAC EFIAWLVKGG GLP-1(7-35;  GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD A8S-K26C-40 1(A8S- AQQTEAHLEI REDGTVGGAA DQSPESLLQL kDa K26C)- KALKPGVIQI LGVKTSRFLC QRPDGALYGS branched L10- LHFDPEACSF RELLLEDGYN VYQSEAHGLP PEG)- FGF21- LHLPGNKSPH RDPAPRGPAR FLPLPGLPPA GSGGGGSGGG- 40KPEGb LPEPPGILAP QPPDVGSSDP LSMVGPSQGR FGF21(33- SPSYAS 209) 75 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7-35;  GLP- TCTAGCTACC TGGAAGGCCA GGCTGCGTGT A8S-K26C)- 1(A8S- GAATTCATCG CGTGGCTGGT TAAAGGCGGT GSGGGGSGGG- K26C)- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21(33- L10- TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG 209) FGF21 GTGCGTCAGC GTTATCTGTA TACCGATGAT GCGCAGCAGA CCGAAGCGCA TCTGGAAATT CGTGAAGATG GCACCGTGGG CGGTGCGGCG GATCAGAGCC CGGAAAGCCT GCTGCAGCTG AAAGCGCTGA AACCGGGCGT GATTCAGATT CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGCGTCCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACAA AAGCCCGCAT CGTGATCCGG CACCGCGTGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGTCTATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 76 V264 HSEGTFTSDV SSYLEGQAAK CFIAWLVKGG GLP-1(7-35;  GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD A8S-E27C-40 1(A8S- AQQTEAHLEI REDGTVGGAA DQSPESLLQL kDa E27C)- KALKPGVIQI LGVKTSRFLC QRPDGALYGS branched 40KPEGb LHFDPEACSF RELLLEDGYN VYQSEAHGLP PEG)- -L10- LHLPGNKSPH RDPAPRGPAR FLPLPGLPPA GSGGGGSGGG- FGF21 LPEPPGILAP QPPDVGSSDP LSMVGPSQGR FGF21(33- SPSYAS 209) 77 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7-35;  GLP- TCTAGCTACC TGGAAGGCCA GGCTGCGAAA A8S-E27C)- 1(A8S- TGTTTCATCG CGTGGCTGGT TAAAGGCGGT GSGGGGSGGG- E27C)- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21(33- L10- TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG 209) FGF21 GTGCGTCAGC GTTATCTGTA TACCGATGAT GCGCAGCAGA CCGAAGCGCA TCTGGAAATT CGTGAAGATG GCACCGTGGG CGGTGCGGCG GATCAGAGCC CGGAAAGCCT GCTGCAGCTG AAAGCGCTGA AACCGGGCGT GATTCAGATT CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGCGTCCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACAA AAGCCCGCAT CGTGATCCGG CACCGCGTGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGTCTATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 78 V244 HSEGTFTSDV SSYLEGQAAK EFIAWLVCGG GLP-1(7-35;  GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD A8S-K34C-40 1(A8S- AQQTEAHLEI REDGTVGGAA DQSPESLLQL kDa K34C)- KALKPGVIQI LGVKTSRFLC QRPDGALYGS branched L10- LHFDPEACSF RELLLEDGYN VYQSEAHGLP PEG)- FGF21- LHLPGNKSPH RDPAPRGPAR FLPLPGLPPA GSGGGGSGGG- 40KPEGb LPEPPGILAP QPPDVGSSDP LSMVGPSQGR FGF21(33- SPSYAS 209) 79 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7-35;  GLP-1(A8S-  TCTAGCTACC TGGAAGGCCA GGCTGCGAAA A8S-K34C)- K34C)- GAATTCATCG CGTGGCTGGT TTGTGGCGGT GSGGGGSGGG- L10-FGF21 TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21(33- TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG 209) GTGCGTCAGC GTTATCTGTA TACCGATGAT GCGCAGCAGA CCGAAGCGCA TCTGGAAATT CGTGAAGATG GCACCGTGGG CGGTGCGGCG GATCAGAGCC CGGAAAGCCT GCTGCAGCTG AAAGCGCTGA AACCGGGCGT GATTCAGATT CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGCGTCCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACAA AAGCCCGCAT CGTGATCCGG CACCGCGTGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGTCTATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 80 V250 HSEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7-35;  GLP- SGGGGCGGGD SSPLLQFGGQ VRQRYLYTDD A8S)- 1(A8S)- AQQTEAHLEI REDGTVGGAA DQSPESLLQL GSGGGGC(40 L10-C- KALKPGVIQI LGVKTSRFLC QRPDGALYGS kDa FGF2140 LHFDPEACSF RELLLEDGYN VYQSEAHGLP branched KPEGb LHLPGNKSPH RDPAPRGPAR FLPLPGLPPA PEG)GGG- LPEPPGILAP QPPDVGSSDP LSMVGPSQGR FGF21(33- SPSYAS 209) 81 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7-35;  GLP- TCTAGCTACC TGGAAGGCCA GGCTGCGAAA A8S)- 1(A8S)- GAATTCATCG CGTGGCTGGT TAAAGGCGGT GSGGGGCGGG- L10-C- TCTGGTGGTG GTGGTTGTGG CGGTGGCGAC FGF21(33- FGF21 TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG 209) GTGCGTCAGC GTTATCTGTA TACCGATGAT GCGCAGCAGA CCGAAGCGCA TCTGGAAATT CGTGAAGATG GCACCGTGGG CGGTGCGGCG GATCAGAGCC CGGAAAGCCT GCTGCAGCTG AAAGCGCTGA AACCGGGCGT GATTCAGATT CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGCGTCCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACAA AAGCCCGCAT CGTGATCCGG CACCGCGTGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGTCTATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 82 V240 HAEGTFTSDV SSYLEGQAAC EFIAWLVKGG GLP-1(7-35;  GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD K26C-40 kDa 1(K26C)- AQQTEAHLEI REDGTVGGAA DQSPESLLQL branched L10- KALKPGVIQI LGVKTSRFLC QRPDGALYGS PEG)- FGF21- LHFDPEACSF RELLLEDGYN VYQSEAHGLP GSGGGGSGGG- 40KPEGb LHLPGNKSPH RDPAPRGPAR FLPLPGLPPA FGF21(33- LPEPPGILAP QPPDVGSSDP LSMVGPSQGR 209) SPSYAS 83 CATGCGGAAG GCACTTTTAC TAGCGATGTT GLP-1(7-35;  GLP- TCTAGCTACC TGGAAGGCCA GGCTGCGTGT K26C)- 1(K26C) GAATTCATCG CGTGGCTGGT TAAAGGCGGT GSGGGGSGGG- -L10- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21(33- FGF21 TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG 209) GTGCGTCAGC GTTATCTGTA TACCGATGAT GCGCAGCAGA CCGAAGCGCA TCTGGAAATT CGTGAAGATG GCACCGTGGG CGGTGCGGCG GATCAGAGCC CGGAAAGCCT GCTGCAGCTG AAAGCGCTGA AACCGGGCGT GATTCAGATT CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGCGTCCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACAA AAGCCCGCAT CGTGATCCGG CACCGCGTGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGTCTATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 84 V241 HAEGTFTSDV SSYLEGQAAK EFIAWLVCGG GLP-1(7-35;  GLP-1- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD K34C-40 kDa (K34C)- AQQTEAHLEI REDGTVGGAA DQSPESLLQL branched L10- KALKPGVIQI LGVKTSRFLC QRPDGALYGS PEG)- 5G521- LHFDPEACSF RELLLEDGYN VYQSEAHGLP GSGGGGSGGG- 40KPEGb LHLPGNKSPH RDPAPRGPAR FLPLPGLPPA FGF21(33- LPEPPGILAP QPPDVGSSDP LSMVGPSQGR 209) SPSYAS 85 CATGCGGAAG GCACTTTTAC TAGCGATGTT GLP-1(7-35;  GLP- TCTAGCTACC TGGAAGGCCA GGCTGCGAAA K34C)- 1(K34C)- GAATTCATCG CGTGGCTGGT TTGTGGCGGT GSGGGGSGGG- L10- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21(33- FGF21 TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG 209) GTGCGTCAGC GTTATCTGTA TACCGATGAT GCGCAGCAGA CCGAAGCGCA TCTGGAAATT CGTGAAGATG GCACCGTGGG CGGTGCGGCG GATCAGAGCC CGGAAAGCCT GCTGCAGCTG AAAGCGCTGA AACCGGGCGT GATTCAGATT CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGCGTCCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACAA AAGCCCGCAT CGTGATCCGG CACCGCGTGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGTCTATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 86 V242 HAEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7- GLP-1- SGGGGCGGGD SSPLLQFGGQ VRQRYLYTDD 35)- L10-C- AQQTEAHLEI REDGTVGGAA DQSPESLLQL GSGGGGC(40 FGF21- KALKPGVIQI LGVKTSRFLC QRPDGALYGS kDa 40KPEGb LHFDPEACSF RELLLEDGYN VYQSEAHGLP branched LHLPGNKSPH RDPAPRGPAR FLPLPGLPPA PEG)GGG- LPEPPGILAP QPPDVGSSDP LSMVGPSQGR FGF21(33- SPSYAS 209) 87 CATGCGGAAG GCACTTTTAC TAGCGATGTT GLP-1(7- GLP-1- TCTAGCTACC TGGAAGGCCA GGCTGCGAAA 35)- L10-C- GAATTCATCG CGTGGCTGGT TAAAGGCGGT GSGGGGCGGG- FGF21 TCTGGTGGTG GTGGTTGTGG CGGTGGCGAC FGF21(33- TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG 209) GTGCGTCAGC GTTATCTGTA TACCGATGAT GCGCAGCAGA CCGAAGCGCA TCTGGAAATT CGTGAAGATG GCACCGTGGG CGGTGCGGCG GATCAGAGCC CGGAAAGCCT GCTGCAGCTG AAAGCGCTGA AACCGGGCGT GATTCAGATT CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGCGTCCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACAA AAGCCCGCAT CGTGATCCGG CACCGCGTGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGTCTATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 88 V267 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD 30)- 30)-L9- AQQTEAHLEI REDGTVGGAA DQSPESLLQL SGGGGSGGG- FGF21 KALKPGVIQI LGVKTSRFLC QRPDGALYGS FGF21(33- (154C)- LHFDPEACSF RELLLEDGYN VYQSEAHGLP 209; R154C- 40KPEGb LHLPGNKSPH CDPAPRGPAR FLPLPGLPPA 40 kDa LPEPPGILAP QPPDVGSSDP LSMVGPSQGR branched SPSYAS PEG) 89 CATGGTGAGG GTACGTTTAC TTCTGATCTG Exendin4(1- Ex(1- TCTAAACAGA TGGAAGAAGA AGCTGTTCGC 30)- 30)-L9- CTGTTCATTG AATGGCTGAA AAATGGTGGT SGGGGSGGG- FGF21 TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21(33- (154C) TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG 209; R154C) GTGCGTCAGC GTTATCTGTA TACCGATGAT GCGCAGCAGA CCGAAGCGCA TCTGGAAATT CGTGAAGATG GCACCGTGGG CGGTGCGGCG GATCAGAGCC CGGAAAGCCT GCTGCAGCTG AAAGCGCTGA AACCGGGCGT GATTCAGATT CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGCGTCCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACAA AAGCCCGCAT TGCGATCCGG CACCGCGTGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGTCTATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 90 V268 HGEGTFTSDL SKQMEGQAVR LFIEWLKNGG Exendin4(1- Ex(1- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD 30; E16G- 30; GQ)- AQQTEAHLEI REDGTVGGAA DQSPESLLQL E17Q)- L9- KALKPGVIQI LGVKTSRFLC QRPDGALYGS SGGGGSGGG- FGF21 LHFDPEACSF RELLLEDGYN VYQSEAHGLP FGF21(33- (154C)- LHLPGNKSPH CDPAPRGPAR FLPLPGLPPA 209; R154C- 40KPEGb LPEPPGILAP QPPDVGSSDP LSMVGPSQGR 40 kDa SPSYAS branched PEG) 91 CATGGTGAGG GTACGTTTAC TTCTGATCTG Exendin4(1- Ex(1- TCTAAACAGA TGGAAGGGCA AGCTGTTCGC 30; E16G- 30; GQ)- CTGTTCATTG AATGGCTGAA AAATGGTGGT E17Q)- L9- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC SGGGGSGGG- FGF21 TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG FGF21(33- (154C) GTGCGTCAGC GTTATCTGTA TACCGATGAT 209; R154C) GCGCAGCAGA CCGAAGCGCA TCTGGAAATT CGTGAAGATG GCACCGTGGG CGGTGCGGCG GATCAGAGCC CGGAAAGCCT GCTGCAGCTG AAAGCGCTGA AACCGGGCGT GATTCAGATT CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGCGTCCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACAA AAGCCCGCAT TGCGATCCGG CACCGCGTGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGTCTATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 92 V301 HSEGTFTSDV SSYLEGQAAK EFIAWLVCGG GLP-1(7-35;  GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD A8S-K34C)- 1(A8S- AQQTEAHLEI REDGTVGGAA DQSPESLLQL GSGGGGSGGG- K34C)- KALKPGVIQI LGVKTSRFLC QRPDGALYGS FGF21(33- FGF21 LHFDPEACSF RELLLEDGYN VYQSEAHGLP 209; R154Pcl) (154Pcl)- LHLPGNKSPH UDPAPRGPAR FLPLPGLPPA LPEPPGILAP QPPDVGSSDP LSMVGPSQGR SPSYAS 93 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7-35;  GLP- TCTAGCTACC TGGAAGGCCA GGCTGCGAAA A8S-K34C)- 1(A8S- GAATTCATCG CGTGGCTGGT TTGTGGCGGT GSGGGGSGGG- K34C)- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21(33- L10- TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG 209; R154TAG) FGF21 GTGCGTCAGC GTTATCTGTA TACCGATGAT (154TAG) GCGCAGCAGA CCGAAGCGCA TCTGGAAATT CGTGAAGATG GCACCGTGGG CGGTGCGGCG GATCAGAGCC CGGAAAGCCT GCTGCAGCTG AAAGCGCTGA AACCGGGCGT GATTCAGATT CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGCGTCCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACAA AAGCCCGCAT TAGGATCCGG CACCGCGTGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGTCTATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 94 V302 HAEGTFTSDV SSYLEGQAAK EFIAWLVCGG GLP-1(7-35;  GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD K34C)- 1(K34C)- AQQTEAHLEI REDGTVGGAA DQSPESLLQL GSGGGGSGGG- L10- KALKPGVIQI LGVKTSRFLC QRPDGALYGS FGF21(33- FGF21 LHFDPEACSF RELLLEDGYN VYQSEAHGLP 209; R154Pcl) (154Pcl) LHLPGNKSPH UDPAPRGPAR FLPLPGLPPA LPEPPGILAP QPPDVGSSDP LSMVGPSQGR SPSYAS 95 CATGCGGAAG GCACTTTTAC TAGCGATGTT GLP-1(7-35;  GLP- TCTAGCTACC TGGAAGGCCA GGCTGCGAAA K34C)- 1(K34C)- GAATTCATCG CGTGGCTGGT TTGTGGCGGT GSGGGGSGGG- L10- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21(33- FGF21 TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG 209; R154TAG) (154TAG) GTGCGTCAGC GTTATCTGTA TACCGATGAT GCGCAGCAGA CCGAAGCGCA TCTGGAAATT CGTGAAGATG GCACCGTGGG CGGTGCGGCG GATCAGAGCC CGGAAAGCCT GCTGCAGCTG AAAGCGCTGA AACCGGGCGT GATTCAGATT CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGCGTCCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACAA AAGCCCGCAT TAGGATCCGG CACCGCGTGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGTCTATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 96 V303 HSEGTFTSDV SSYLEGQAAK EFIAWLVUGG GLP-1(7-35;  GLP-1(A8S- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD A8S- K34Pcl)- AQQTEAHLEI REDGTVGGAA DQSPESLLQL K34Pcl)- L10-FGF21 KALKPGVIQI LGVKTSRFLC QRPDGALYGS GSGGGGSGGG- (154C) LHFDPEACSF RELLLEDGYN VYQSEAHGLP FGF21(33- LHLPGNKSPH CDPAPRGPAR FLPLPGLPPA 209; R154C) LPEPPGILAP QPPDVGSSDP LSMVGPSQGR SPSYAS 97 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7-35;  GLP-1(A8S- TCTAGCTACC TGGAAGGCCA GGCTGCGAAA A8S- K34TAG)- GAATTCATCG CGTGGCTGGT TTAGGGCGGT K34TAG)- L10-FGF21 TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC GSGGGGSGGG- (154C) TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG FGF21(33- GTGCGTCAGC GTTATCTGTA TACCGATGAT 209; R154C) GCGCAGCAGA CCGAAGCGCA TCTGGAAATT CGTGAAGATG GCACCGTGGG CGGTGCGGCG GATCAGAGCC CGGAAAGCCT GCTGCAGCTG AAAGCGCTGA AACCGGGCGT GATTCAGATT CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGCGTCCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACAA AAGCCCGCAT TGCGATCCGG CACCGCGTGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGTCTATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 98 V281 HSEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7- GLP-1 SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD 35; A8S)- (A8S)- AQETEAHLEI REDGTVGGAA HQSPESLLEL GSGGGGSGGG- L10- KALKPGVIQI LGVKTSRFLC QKPDGALYGS FGF21(33- FGF21 LHFDPEACSF RELLLEDGYN VYQSEAHGLP 209; Q56E- (V76- LHLPGNRSPH RDPAPQGPAR FLPLPGLPPA D74H-Q82E- 154R) LPEPPGILAP QPPDVGSSDP LAMVGPSQGR R105K- SPSYAS K150R- R159Q- S195A) 99 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7- GLP- TCTAGCTACC TGGAAGGCCA GGCTGCGAAA 35; A8S)- 1(A8S)- GAATTCATCG CGTGGCTGGT TAAAGGCGGT GSGGGGSGGG- L10- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21(33- FGF21 TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG 209; Q56E- (V76- GTGCGTCAGC GTTATCTGTA TACCGATGAT D74H-Q82E- 154R) GCGCAGGAAA CCGAAGCGCA TCTGGAAATT R105K- CGTGAAGATG GCACCGTGGG CGGTGCGGCG K150R- CATCAGAGCC CGGAAAGCCT GCTGGAACTG R159Q- AAAGCGCTGA AACCGGGCGT GATTCAGATT S195A) CTGGGCGTGA AAACCAGCCG TTTTCTGTGC CAGAAACCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACCG TAGCCCGCAT CGTGATCCGG CACCGCAGGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGGCGATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 100 V304 HSEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7- GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD 35; A8S)- 1(A8S)- AQETEAHLEI REDGTVGGAA HQSPESLLEL GSGGGGSGGG- L10- KALKPGVIQI LGVKTSRFLC QKPDGALYGS FGF21(33- FGF21 LHFDPEACSF RELLLEEGYN VYQSEAHGLP 209; Q56E- (V76- LHLPGNRSPH RDPAPQGPAR FLPLPGLPPA D74H-Q82E- 154R- LPEPPGILAP QPPDVGSSDP LAMVGPSQGR R105K- 130E) SPSYAS D130E- K150R- R159Q- S195A) 101 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7- GLP- TCTAGCTACC TGGAAGGCCA GGCTGCGAAA 35; A8S)- 1(A8S)- GAATTCATCG CGTGGCTGGT TAAAGGCGGT GSGGGGSGGG- L10- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21(33- FGF21 TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG 209; Q56E- (V76- GTGCGTCAGC GTTATCTGTA TACCGATGAT D74H-Q82E- 154R- GCGCAGGAAA CCGAAGCGCA TCTGGAAATT R105K- 130E) CGTGAAGATG GCACCGTGGG CGGTGCGGCG D130E- CATCAGAGCC CGGAAAGCCT GCTGGAACTG K150R- AAAGCGCTGA AACCGGGCGT GATTCAGATT R159Q- CTGGGCGTGA AAACCAGCCG TTTTCTGTGC S195A) CAGAAACCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA AGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACCG TAGCCCGCAT CGTGATCCGG CACCGCAGGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGGCGATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 102 V273 HSEGTFTSDV SSYLEGQAAK EFIAWLVCGG GLP-1(7- GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD 35; A8S- 1(A8S- AQETEAHLEI REDGTVGGAA HQSPESLLEL K34C-40 kDa K34C)- KALKPGVIQI LGVKTSRFLC QKPDGALYGS branched L10- LHFDPEACSF RELLLEEGYN VYQSEAHGLP PEG)- FGF21 LHLPGNRSPH RDPAPQGPAR FLPLPGLPPA GSGGGGSGGG- (V76- LPEPPGILAP QPPDVGSSDP LAMVGPSQGR FGF21(33- 154R)- SPSYAS 209; Q56E- 40KPEGb D74H-Q82E- R105K- D130E- K150R- R159Q- S195A) 103 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7- GLP- TCTAGCTACC TGGAAGGCCA GGCTGCGAAA 35; A8S- 1(A8S- GAATTCATCG CGTGGCTGGT TTGTGGCGGT K34C)- K34C)- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC GSGGGGSGGG- L10- TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG FGF21(33- FGF21 GTGCGTCAGC GTTATCTGTA TACCGATGAT 209; Q56E- (V76- GCGCAGGAAA CCGAAGCGCA TCTGGAAATT D74H-Q82E- 154R) CGTGAAGATG GCACCGTGGG CGGTGCGGCG R105K- CATCAGAGCC CGGAAAGCCT GCTGGAACTG D130E- AAAGCGCTGA AACCGGGCGT GATTCAGATT K150R- CTGGGCGTGA AAACCAGCCG TTTTCTGTGC R159Q- CAGAAACCGG ATGGCGCGCT GTATGGCAGC S195A) CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA AGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACCG TAGCCCGCAT CGTGATCCGG CACCGCAGGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGGCGATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 104 V305 HSEGTFTSDV SSYLEGQAAK EFIAWLVCGG GLP-1(7- GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD 35; A8S- 1(A8S- AQETEAHLEI REDGTVGGAA HQSPESLLEL K34C-40 kDa K34C)- KALKPGVIQI LGVKTSRFLC QKPDGALYGS branched L10- LHFDPEACSF RELLLEDGYN VYQSEAHGLP PEG)- FGF21 LHLPGNRSPH RDPAPQGPAR FLPLPGLPPA GSGGGGSGGG- (V76- LPEPPGILAP QPPDVGSSDP LAMVGPSQAR FGF21(33- 154R- SPSYAS 209; Q56E- 202A)- D74H-Q82E- 40KPEGb R105K- K150R- R159Q- S195A- G202A) 105 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7- GLP-1 TCTAGCTACC TGGAAGGCCA GGCTGCGAAA 35; A8S- (A8S- GAATTCATCG CGTGGCTGGT TTGTGGCGGT K34C)- K34C)- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC GSGGGGSGGG- L10- TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG FGF21(33- FGF21 GTGCGTCAGC GTTATCTGTA TACCGATGAT 209; Q56E- (V76- GCGCAGGAAA CCGAAGCGCA TCTGGAAATT D74H-Q82E- 154R- CGTGAAGATG GCACCGTGGG CGGTGCGGCG R105K- 202A) CATCAGAGCC CGGAAAGCCT GCTGGAACTG K150R- AAAGCGCTGA AACCGGGCGT GATTCAGATT R159Q- CTGGGCGTGA AAACCAGCCG TTTTCTGTGC S195A- CAGAAACCGG ATGGCGCGCT GTATGGCAGC G202A) CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACCG TAGCCCGCAT CGTGATCCGG CACCGCAGGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGGCGATGG TGGGTCCGAG CCAGGCGCGT AGCCCGAGCT ATGCGAGCTA A 108 V306 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1-30)- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD 30)- L9-FGF21 AQETEAHLEI REDGTVGGAA HQSPESLLEL SGGGGSGGG- (V76- KALKPGVIQI LGVKTSRFLC QKPDGALYGS FGF21(33- 154R) LHFDPEACSF RELLLEEGYN VYQSEAHGLP 209; Q56E- LHLPGNRSPH RDPAPQGPAR FLPLPGLPPA D74H-Q82E- LPEPPGILAP QPPDVGSSDP LAMVGPSQGR R105K- SPSYAS D130E- K150R- R159Q- S195A) 109 CATGGTGAGG GTACGTTTAC TTCTGATCTG Exendin4(1- Ex(1-30)-  TCTAAACAGA TGGAAGAAGA AGCTGTTCGC 30)- L9-FGF21 CTGTTCATTG AATGGCTGAA AAATGGTGGT SGGGGSGGG- (V76- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21(33- 154R) TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG 209; Q56E- GTGCGTCAGC GTTATCTGTA TACCGATGAT D74H-Q82E- GCGCAGGAAA CCGAAGCGCA TCTGGAAATT R105K- CGTGAAGATG GCACCGTGGG CGGTGCGGCG D130E- CATCAGAGCC CGGAAAGCCT GCTGGAACTG K150R- AAAGCGCTGA AACCGGGCGT GATTCAGATT R159Q- CTGGGCGTGA AAACCAGCCG TTTTCTGTGC S195A) CAGAAACCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA AGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACCG TAGCCCGCAT CGTGATCCGG CACCGCAGGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGGCGATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 110 V274 HGEGTFTSDL SKQMEEEAVR LFIEWLCNGG Exendin4(1- Ex(1- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD 30; K27C-40 30; K27C)- AQETEAHLEI REDGTVGGAA HQSPESLLEL kDa L9- KALKPGVIQI LGVKTSRFLC QKPDGALYGS branched FGF21 LHFDPEACSF RELLLEEGYN VYQSEAHGLP PEG)- (V76- LHLPGNRSPH RDPAPQGPAR FLPLPGLPPA SGGGGSGGG- 154R- LPEPPGILAP QPPDVGSSDP LAMVGPSQGR FGF21(33- 130E)- SPSYAS 209; Q56E- 40KPEGb D74H-Q82E- R105K- D130E- K150R- R159Q- S195A) 111 CATGGTGAGG GTACGTTTAC TTCTGATCTG Exendin4(1- Ex(1-30;  TCTAAACAGA TGGAAGAAGA AGCTGTTCGC 30; K27C)- K27C)- CTGTTCATTG AATGGCTGTG TAATGGTGGT SGGGGSGGG- L9- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21(33- FGF21 TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG 209; Q56E- (V76- GTGCGTCAGC GTTATCTGTA TACCGATGAT D74H-Q82E- 154R- GCGCAGGAAA CCGAAGCGCA TCTGGAAATT R105K- 130E) CGTGAAGATG GCACCGTGGG CGGTGCGGCG D130E- CATCAGAGCC CGGAAAGCCT GCTGGAACTG K150R- AAAGCGCTGA AACCGGGCGT GATTCAGATT R159Q- CTGGGCGTGA AAACCAGCCG TTTTCTGTGC S195A) CAGAAACCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA AGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACCG TAGCCCGCAT CGTGATCCGG CACCGCAGGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGGCGATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 112 V307 HGEGTFTSDL SKQMEEEAVR LFIEWLKCGG Exendin4(1- Ex(1-30;  SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD 30; N28C-40 N28C)- AQETEAHLEI REDGTVGGAA HQSPESLLEL kDa L9- KALKPGVIQI LGVKTSRFLC QKPDGALYGS branched FGF21 LHFDPEACSF RELLLEEGYN VYQSEAHGLP PEG)- (V76- LHLPGNRSPH RDPAPQGPAR FLPLPGLPPA SGGGGSGGG- 154R- LPEPPGILAP QPPDVGSSDP LAMVGPSQGR FGF21(33- 130E)- SPSYAS 209; Q56E- 40KPEGb D74H-Q82E- R105K- D130E- K150R- R159Q- S195A) 113 CATGGTGAGG GTACGTTTAC TTCTGATCTG Exendin4(1- Ex(1-30;  TCTAAACAGA TGGAAGAAGA AGCTGTTCGC 30; N28C)- N28C)- CTGTTCATTG AATGGCTGAA ATGTGGTGGT SGGGGSGGG- L9- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21(33- FGF21 TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG 209; Q56E- (V76- GTGCGTCAGC GTTATCTGTA TACCGATGAT D74H-Q82E- 154R- GCGCAGGAAA CCGAAGCGCA TCTGGAAATT R105K- 130E) CGTGAAGATG GCACCGTGGG CGGTGCGGCG D130E- CATCAGAGCC CGGAAAGCCT GCTGGAACTG K150R- AAAGCGCTGA AACCGGGCGT GATTCAGATT R159Q- CTGGGCGTGA AAACCAGCCG TTTTCTGTGC S195A) CAGAAACCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA AGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACCG TAGCCCGCAT CGTGATCCGG CACCGCAGGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGGCGATGG TGGGTCCGAG CCAGGGTCGT AGCCCGAGCT ATGCGAGCTA A 114 V308 HGEGTFTSDL SKQMEEEAVR LFIEWLCNGG Exendin4(1- Ex(1- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD 30; K27C-40 30;  AQETEAHLEI REDGTVGGAA HQSPESLLEL kDa K27C)-- KALKPGVIQI LGVKTSRFLC QKPDGALYGS branched L9-FGF21 LHFDPEACSF RELLLEDGYN VYQSEAHGLP PEG)- (V76- LHLPGNRSPH RDPAPQGPAR FLPLPGLPPA SGGGGSGGG- 154R- LPEPPGILAP QPPDVGSSDP LAMVGPSQAR FGF21(33- 202A)- SPSYAS 209; Q56E- 40KPEGb D74H-Q82E- R105K- K150R- R159Q- S195A- G202A) 115 CATGGTGAGG GTACGTTTAC TTCTGATCTG Exendin4(1- Ex(1-30;  TCTAAACAGA TGGAAGAAGA AGCTGTTCGC 30; K27C)- K27C)- CTGTTCATTG AATGGCTGTG TAATGGTGGT SGGGGSGGG- L9- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21(33- FGF21 TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG 209; Q56E- (V76- GTGCGTCAGC GTTATCTGTA TACCGATGAT D74H-Q82E- 154R- GCGCAGGAAA CCGAAGCGCA TCTGGAAATT R105K- 202A) CGTGAAGATG GCACCGTGGG CGGTGCGGCG K150R- CATCAGAGCC CGGAAAGCCT GCTGGAACTG R159Q- AAAGCGCTGA AACCGGGCGT GATTCAGATT S195A- CTGGGCGTGA AAACCAGCCG TTTTCTGTGC G202A) CAGAAACCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACCG TAGCCCGCAT CGTGATCCGG CACCGCAGGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGGCGATGG TGGGTCCGAG CCAGGCGCGT AGCCCGAGCT ATGCGAGCTA A 116 V309 HGEGTFTSDL SKQMEEEAVR LFIEWLKCGG Exendin4(1- Ex(1-30;  SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD 30; N28C-40 N28C)- AQETEAHLEI REDGTVGGAA HQSPESLLEL kDa L9-FGF21 KALKPGVIQI LGVKTSRFLC QKPDGALYGS branched (V76-154R- LHFDPEACSF RELLLEDGYN VYQSEAHGLP PEG)- 202A)- LHLPGNRSPH RDPAPQGPAR FLPLPGLPPA SGGGGSGGG- 40KPEGb LPEPPGILAP QPPDVGSSDP LAMVGPSQAR FGF21(33- SPSYAS 209; Q56E- D74H-Q82E- R105K- K150R- R159Q- S195A- G202A) 117 CATGGTGAGG GTACGTTTAC TTCTGATCTG Exendin4(1- Ex(1-30;  TCTAAACAGA TGGAAGAAGA AGCTGTTCGC 30; N28C)- N28C)- CTGTTCATTG AATGGCTGAA ATGTGGTGGT SGGGGSGGG- L9-FGF21 TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21(33- (V76- TCGAGCCCGC TGCTGCAGTT TGGCGGCCAG 209; Q56E- 154R-202A) GTGCGTCAGC GTTATCTGTA TACCGATGAT D74H-Q82E- GCGCAGGAAA CCGAAGCGCA TCTGGAAATT R105K- CGTGAAGATG GCACCGTGGG CGGTGCGGCG K150R- CATCAGAGCC CGGAAAGCCT GCTGGAACTG R159Q- AAAGCGCTGA AACCGGGCGT GATTCAGATT S195A- CTGGGCGTGA AAACCAGCCG TTTTCTGTGC G202A) CAGAAACCGG ATGGCGCGCT GTATGGCAGC CTGCATTTTG ATCCGGAAGC GTGCAGCTTT CGTGAACTGC TGCTGGAAGA TGGCTATAAC GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACCG TAGCCCGCAT CGTGATCCGG CACCGCAGGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGGCGATGG TGGGTCCGAG CCAGGCGCGT AGCCCGAGCT ATGCGAGCTA A 118 V276 HSEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7- GLP- GSGGGGSGGG GSGGGGSGGG DSSPLLQFGG 35; A8S)- 1(A8S)- QVRQRYLYTD DAQETEAHLE IREDGTVGGA GGS(GGGGS)₃- L20- AHQSPESLLE LKALKPGVIQ ILGVKTSRFL FGF21(33- FGF21 CQKPDGALYG SLHFDPEACS FRELLLEDGY 209; Q56E- (V76)- NVYQSEAHGL PLHLPGNRSP HCDPAPQGPA D74H-Q82E- 40KPEGb RFLPLPGLPP ALPEPPGILA PQPPDVGSSD R105K- PLAMVGPSQG RSPSYAS K150R- R154C-40 kDa branched PEG-R159Q- S195A) 119 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7- GLP-1 TCTAGCTACC TGGAAGGCCA GGCTGCGAAA 35; A8S)- (A8S)-L20- GAATTCATCG CGTGGCTGGT TAAAGGCGGT GGS(GGGGS)₃- FGF21 GGTAGCGGTG GCGGCGGTTC TGGTGGTGGT FGF21(33- (V76) GGTTCTGGCG GTGGCGGTAG CGGTGGCGGC 209; Q56E- GATAGCAGCC CGCTGCTGCA GTTTGGCGGC D74H-Q82E- CAGGTGCGTC AGCGTTATCT GTATACCGAT R105K- GATGCGCAGG AAACCGAAGC GCATCTGGAA K150R- ATTCGTGAAG ATGGCACCGT GGGCGGTGCG R154C- GCGCATCAGA GCCCGGAAAG CCTGCTGGAA R159Q- CTGAAAGCGC TGAAACCGGG CGTGATTCAG S195A) ATTCTGGGCG TGAAAACCAG CCGTTTTCTG TGCCAGAAAC CGGATGGCGC GCTGTATGGC AGCCTGCATT TTGATCCGGA AGCGTGCAGC TTTCGTGAAC TGCTGCTGGA AGATGGCTAT AACGTGTATC AGAGCGAAGC GCATGGCCTG CCGCTGCATC TGCCGGGCAA CCGTAGCCCG CATTGCGATC CGGCACCGCA GGGTCCGGCG CGTTTTCTGC CGCTGCCGGG TCTGCCGCCG GCACTGCCGG AACCGCCGGG TATTCTGGCC CCGCAGCCGC CGGATGTTGG TAGCAGCGAT CCGCTGGCGA TGGTGGGTCC GAGCCAGGGT CGTAGCCCGA GCTATGCGAG CTAA 120 V197 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Exendin GGGGSDKTHT CPPCPAPEAA GGPSVFLFPP 30)-(GGGGS)- (1-30)- KPKDTLMISR TPEVTCVVVD VSHEDPEVKF Fc-(GGGGS)₃- L5-Fc- NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV FGF21(33- L15- LTVLHQDWLN GKEYKCKVSN KALPAPIEKT 209; Q55C- FGF21 ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL R105K- (V103)) TCLVKGFYPS DIAVEWESNG QPENNYKTTP G148C- PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC K150R- SVMHEALHNH YTQKSLSLSP GKGGGGSGGG P158S- GSGGGGSDSS PLLQFGGQVR QRYLYTDDAC S195A- QTEAHLEIRE DGTVGGAADQ SPESLLQLKA P199G- LKPGVIQILG VKTSRFLCQK PDGALYGSLH G202A) FDPEACSFRE LLLEDGYNVY QSEAHGLPLH LPCNRSPHRD PASRGPARFL PLPGLPPALP EPPGILAPQP PDVGSSDPLA MVGGSQARSP SYAS 121 V196 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1- GGGGSGGGGS GGGGSDKTHT CPPCPAPEAA 30)-(GGGGS)₃- 30)-L15- GGPSVFLFPP KPKDTLMISR TPEVTCVVVD Fc-(GGGGS)₃- Fc-L15- VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ FGF21(33- FGF21 YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN 209; Q55C- (V103) KALPAPIEKT ISKAKGQPRE PQVYTLPPSR R105K- EEMTKNQVSL TCLVKGFYPS DIAVEWESNG G148C- QPENNYKTTP PVLDSDGSFF LYSKLTVDKS K150R- RWQQGNVFSC SVMHEALHNH YTQKSLSLSP P158S- GKGGGGSGGG GSGGGGSDSS PLLQFGGQVR S195A- QRYLYTDDAC QTEAHLEIRE DGTVGGAADQ P199G- SPESLLQLKA LKPGVIQILG VKTSRFLCQK G202A) PDGALYGSLH FDPEACSFRE LLLEDGYNVY QSEAHGLPLH LPCNRSPHRD PASRGPARFL PLPGLPPALP EPPGILAPQP PDVGSSDPLA MVGGSQARSP SYAS 122 V199 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1- PSSGAPPPSG GGGSDKTHTC PPCPAPEAAG 39)-(GGGGS)- 39)-L5- GPSVFLFPPK PKDTLMISRT PEVTCVVVDV Fc-(GGGGS)₃- Fc-L15- SHEDPEVKFN WYVDGVEVHN AKTKPREEQY FGF21(33- FGF21 NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK 209; Q55C- (V103) ALPAPIEKTI SKAKGQPREP QVYTLPPSRE R105K- EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ G148C- PENNYKTTPP VLDSDGSFFL YSKLTVDKSR K150R- WQQGNVFSCS VMHEALHNHY TQKSLSLSPG P158S- KGGGGSGGGG SGGGGSDSSP LLQFGGQVRQ S195A- RYLYTDDACQ TEAHLEIRED GTVGGAADQS P199G- PESLLQLKAL KPGVIQILGV KTSRFLCQKP G202A) DGALYGSLHF DPEACSFREL LLEDGYNVYQ SEAHGLPLHL PCNRSPHRDP ASRGPARFLP LPGLPPALPE PPGILAPQPP DVGSSDPLAM VGGSQARSPS YAS 123 V198 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1- PSSGAPPPSG GGGSGGGGSG GGGSDKTHTC 39)-(GGGGS)₃- 39)- PPCPAPEAAG GPSVFLFPPK PKDTLMISRT Fc-(GGGGS)₃- L15-Fc- PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN FGF21(33- L15- AKTKPREEQY NSTYRVVSVL TVLHQDWLNG 209; Q55C- FGF21 KEYKCKVSNK ALPAPIEKTI SKAKGQPREP R105K- (V103) QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD G148C- IAVEWESNGQ PENNYKTTPP VLDSDGSFFL K150R- YSKLTVDKSR WQQGNVFSCS VMHEALHNHY P158S- TQKSLSLSPG KGGGGSGGGG SGGGGSDSSP S195A- LLQFGGQVRQ RYLYTDDACQ TEAHLEIRED P199G- GTVGGAADQS PESLLQLKAL KPGVIQILGV G202A) KTSRFLCQKP DGALYGSLHF DPEACSFREL LLEDGYNVYQ SEAHGLPLHL PCNRSPHRDP ASRGPARFLP LPGLPPALPE PPGILAPQPP DVGSSDPLAM VGGSQARSPS YAS 124 V203 HSEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7-35;  GLP- GGGSDKTHTC PPCPAPEAAG GPSVFLFPPK A8S)- 1(A8S)- PKDTLMISRT PEVTCVVVDV SHEDPEVKFN (GGGGS)-Fc- L5-Fc- WYVDGVEVHN AKTKPREEQY NSTYRVVSVL (GGGGS)₃- L15- TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI FGF21(33- FGF21 SKAKGQPREP QVYTLPPSRE EMTKNQVSLT 209; Q55C- (V103) CLVKGFYPSD IAVEWESNGQ PENNYKTTPP R105K- VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS G148C- VMHEALHNHY TQKSLSLSPG KGGGGSGGGG K150R- SGGGGSDSSP LLQFGGQVRQ RYLYTDDACQ P158S- TEAHLEIRED GTVGGAADQS PESLLQLKAL S195A- KPGVIQILGV KTSRFLCQKP DGALYGSLHF P199G- DPEACSFREL LLEDGYNVYQ SEAHGLPLHL G202A) PCNRSPHRDP ASRGPARFLP LPGLPPALPE PPGILAPQPP DVGSSDPLAM VGGSQARSPS YAS 125 V202 HSEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7-35;  GLP- GGGSGGGGSG GGGSDKTHTC PPCPAPEAAG A8S)- 1(A8S)- GPSVFLFPPK PKDTLMISRT PEVTCVVVDV (GGGGS)₃-Fc- L15-Fc- SHEDPEVKFN WYVDGVEVHN AKTKPREEQY (GGGGS)₃- L15- NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK FGF21(33- FGF21 ALPAPIEKTI SKAKGQPREP QVYTLPPSRE 209; Q55C- (V103) EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ R105K- PENNYKTTPP VLDSDGSFFL YSKLTVDKSR G148C- WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K150R- KGGGGSGGGG SGGGGSDSSP LLQFGGQVRQ P158S- RYLYTDDACQ TEAHLEIRED GTVGGAADQS 5195A- PESLLQLKAL KPGVIQILGV KTSRFLCQKP P199G- DGALYGSLHF DPEACSFREL LLEDGYNVYQ G202A) SEAHGLPLHL PCNRSPHRDP ASRGPARFLP LPGLPPALPE PPGILAPQPP DVGSSDPLAM VGGSQARSPS YAS 126 V310 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1- PSSGAPPPSG GGGSGGGGSG GGGSGGGGDS 39)- 39)- SPLLQFGGQV RQRYLYTDDA QETEAHLEIR (GGGGS)₃GGGG L19- EDGTVGGAAH QSPESLLELK ALKPGVIQIL -FGF21(33- FGF21 GVKTSRFLCQ KPDGALYGSL HFDPEACSFR 209; Q56E- (V76)- ELLLEDGYNV YQSEAHGLPL HLPGNRSPHC D74H-Q82E- 40KPEGb DPAPQGPARF LPLPGLPPAL PEPPGILAPQ R105K- PPDVGSSDPL AMVGPSQGRS PSYAS K150R-R154C-40 kDa branched PEG- R159Q- S195A) 127 CATGGTGAGG GTACGTTTAC TTCTGATCTG Exendin4(1- Ex(1- TCTAAACAGA TGGAAGAAGA AGCTGTTCGC 39)- 39)- CTGTTCATTG AATGGCTGAA AAATGGTGGT (GGGGS)₃GGGG- L19- CCGTCCTCCG GCGCTCCTCC GCCTTCTGGT FGF21(33- FGF21 GGTGGTGGTT CTGGCGGTGG CGGTTCTGGC 209; Q56E- (V76) GGCGGTGGTA GCGGTGGCGG CGGTGATAGC D74H-Q82E- AGCCCGCTGC TGCAGTTTGG CGGCCAGGTG R105K- CGTCAGCGTT ATCTGTATAC CGATGATGCG K150R- CAGGAAACCG AAGCGCATCT GGAAATTCGT R154C- GAAGATGGCA CCGTGGGCGG TGCGGCGCAT R159Q- CAGAGCCCGG AAAGCCTGCT GGAACTGAAA S195A) GCGCTGAAAC CGGGCGTGAT TCAGATTCTG GGCGTGAAAA CCAGCCGTTT TCTGTGCCAG AAACCGGATG GCGCGCTGTA TGGCAGCCTG CATTTTGATC CGGAAGCGTG CAGCTTTCGT GAACTGCTGC TGGAAGATGG CTATAACGTG TATCAGAGCG AAGCGCATGG CCTGCCGCTG CATCTGCCGG GCAACCGTAG CCCGCATTGC GATCCGGCAC CGCAGGGTCC GGCGCGTTTT CTGCCGCTGC CGGGTCTGCC GCCGGCACTG CCGGAACCGC CGGGTATTCT GGCCCCGCAG CCGCCGGATG TTGGTAGCAG CGATCCGCTG GCGATGGTGG GTCCGAGCCA GGGTCGTAGC CCGAGCTATG CGAGCTAA 31 V206 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1- PSSGAPPPSD KTHTCPPCPA PEAAGGPSVF 39)-Fc- 39)-L0- LFPPKPKDTL MISRTPEVTC VVVDVSHEDP FGF21(33- Fc-L0- EVKFNWYVDG VEVHNAKTKP REEQYNSTYR 209; Q55C- FGF21 VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP R105K- (V103) IEKTISKAKG QPREPQVYTL PPSREEMTKN G148C- QVSLTCLVKG FYPSDIAVEW ESNGQPENNY K150R- KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN P158S- VFSCSVMHEA LHNHYTQKSL SLSPGKDSSP 5195A- LLQFGGQVRQ RYLYTDDACQ TEAHLEIRED P199G- GTVGGAADQS PESLLQLKAL KPGVIQILGV G202A) KTSRFLCQKP DGALYGSLHF DPEACSFREL LLEDGYNVYQ SEAHGLPLHL PCNRSPHRDP ASRGPARFLP LPGLPPALPE PPGILAPQPP DVGSSDPLAM VGGSQARSPS YAS 32 V208 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1- PSSGAPPPSD KTHTCPPCPA PEAAGGPSVF 39)-Fc-GS- 39)-L0- LFPPKPKDTL MISRTPEVTC VVVDVSHEDP FGF21(33- Fc-L2- EVKFNWYVDG VEVHNAKTKP REEQYNSTYR 209; Q55C- FGF21 VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP A109T- (V101) IEKTISKAKG QPREPQVYTL PPSREEMTKN G148C- QVSLTCLVKG FYPSDIAVEW ESNGQPENNY K150R- KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN P158S- VFSCSVMHEA LHNHYTQKSL SLSPGKGSDS S195A- SPLLQFGGQV RQRYLYTDDA CQTEAHLEIR P199G- EDGTVGGAAD QSPESLLQLK ALKPGVIQIL G202A) GVKTSRFLCQ RPDGTLYGSL HFDPEACSFR ELLLEDGYNV YQSEAHGLPL HLPCNRSPHR DPASRGPARF LPLPGLPPAL PEPPGILAPQ PPDVGSSDPL AMVGGSQARS PSYAS 33 CACGGAGAAG GCACCTTTAC ATCGGACTTG Exendin4(1- Ex(1- TCGAAGCAGA TGGAGGAAGA AGCGGTGAGG 39)-Fc-GS- 39)-L0- CTCTTCATCG AGTGGCTCAA GAATGGAGGA FGF21(33- Fc-L2- CCCTCAAGCG GAGCGCCTCC TCCTTCCGAC 209; Q55C- FGF21 AAAACCCATA CATGTCCGCC TTGTCCCGCA A109T- (V101) CCAGAAGCAG CGGGTGGGCC CTCGGTGTTC G148C- CTGTTCCCGC CAAAACCGAA GGACACACTT K150R- ATGATTTCAC GCACACCGGA AGTGACTTGC P158S- GTCGTGGTGG ATGTATCGCA CGAGGACCCC S195A- GAGGTCAAAT TCAACTGGTA TGTCGATGGA P199G- GTGGAGGTGC ACAATGCAAA GACCAAGCCG G202A) AGGGAAGAAC AATACAATAG CACGTACCGA GTCGTGTCCG TCTTGACGGT CCTTCACCAG GACTGGCTGA ACGGAAAGGA GTACAAGTGC AAAGTGAGCA ATAAGGCCCT CCCTGCCCCG ATTGAGAAAA CCATTTCCAA GGCCAAAGGT CAGCCTAGAG AACCTCAAGT GTATACTCTT CCGCCCTCAC GCGAAGAGAT GACGAAAAAC CAAGTGTCGC TTACGTGTCT TGTCAAAGGT TTCTACCCCT CGGACATCGC CGTAGAGTGG GAGTCGAACG GCCAGCCGGA GAACAACTAC AAGACCACGC CCCCTGTCTT GGATAGCGAC GGATCGTTTT TCCTCTACTC GAAACTCACA GTAGATAAGT CCCGATGGCA ACAGGGTAAT GTCTTTAGCT GCAGCGTGAT GCACGAGGCG CTTCACAATC ATTACACACA AAAATCACTG TCGCTTAGCC CGGGAAAGGG TTCAGATTCG TCGCCCCTGT TGCAGTTTGG TGGACAGGTC AGACAGCGCT ACCTTTACAC GGATGACGCC TGCCAGACAG AGGCACACCT CGAAATCAGA GAGGACGGTA CGGTCGGGGG TGCGGCCGAT CAGAGCCCCG AGTCGCTTCT CCAGTTGAAG GCCCTTAAGC CAGGAGTCAT CCAGATTTTG GGAGTAAAGA CCTCACGGTT TCTCTGTCAG CGTCCAGATG GGACACTGTA CGGCTCATTG CATTTCGATC CCGAAGCGTG CTCGTTCCGG GAGTTGCTGC TTGAGGACGG ATATAACGTC TATCAGAGCG AAGCGCATGG CCTCCCCCTT CACCTCCCGT GTAACAGGTC GCCGCATCGG GATCCGGCCT CGAGGGGTCC CGCGAGATTT CTTCCGTTGC CCGGGTTGCC TCCCGCGCTG CCCGAGCCTC CCGGGATCCT CGCGCCACAG CCTCCTGATG TAGGGTCCTC GGACCCTTTG GCGATGGTAG GTGGATCACA AGCACGGTCC CCGAGCTATG CATCA 36 V209 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1-39)- PSSGAPPPSG GGGSGGGGSG GGGSDKTHTC 39)- L15-Fc-L2- PPCPAPEAAG GPSVFLFPPK PKDTLMISRT (GGGGS)₃- FGF21 PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN Fc-GS- (V101) AKTKPREEQY NSTYRVVSVL TVLHQDWLNG FGF21(33- KEYKCKVSNK ALPAPIEKTI SKAKGQPREP 209; Q55C- QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD A109T- IAVEWESNGQ PENNYKTTPP VLDSDGSFFL G148C- YSKLTVDKSR WQQGNVFSCS VMHEALHNHY K150R- TQKSLSLSPG KGSDSSPLLQ FGGQVRQRYL P158S- YTDDACQTEA HLEIREDGTV GGAADQSPES 5195A- LLQLKALKPG VIQILGVKTS RFLCQRPDGT P199G- LYGSLHFDPE ACSFRELLLE DGYNVYQSEA G202A) HGLPLHLPCN RSPHRDPASR GPARFLPLPG LPPALPEPPG ILAPQPPDVG SSDPLAMVGG SQARSPSYAS 37 CACGGAGAAG GCACCTTTAC ATCGGACTTG Exendin4(1- Ex(1-39)- TCGAAGCAGA TGGAGGAAGA AGCGGTGAGG 39)- L15-Fc-L2- CTCTTCATCG AGTGGCTCAA GAATGGAGGA (GGGGS)₃- FGF21 CCCTCAAGCG GAGCGCCTCC TCCTTCCGGA Fc-GS- (V101) GGAGGTGGGT CGGGCGGTGG AGGCTCCGGA FGF21(33- GGGGGAGGGA GCGACAAAAC CCATACATGT 209; Q55C- CCGCCTTGTC CCGCACCAGA AGCAGCGGGT A109T- GGGCCCTCGG TGTTCCTGTT CCCGCCAAAA G148C- CCGAAGGACA CACTTATGAT TTCACGCACA K150R- CCGGAAGTGA CTTGCGTCGT GGTGGATGTA P158S- TCGCACGAGG ACCCCGAGGT CAAATTCAAC 5195A- TGGTATGTCG ATGGAGTGGA GGTGCACAAT P199G- GCAAAGACCA AGCCGAGGGA AGAACAATAC G202A) AATAGCACGT ACCGAGTCGT GTCCGTCTTG ACGGTCCTTC ACCAGGACTG GCTGAACGGA AAGGAGTACA AGTGCAAAGT GAGCAATAAG GCCCTCCCTG CCCCGATTGA GAAAACCATT TCCAAGGCCA AAGGTCAGCC TAGAGAACCT CAAGTGTATA CTCTTCCGCC CTCACGCGAA GAGATGACGA AAAACCAAGT GTCGCTTACG TGTCTTGTCA AAGGTTTCTA CCCCTCGGAC ATCGCCGTAG AGTGGGAGTC GAACGGCCAG CCGGAGAACA ACTACAAGAC CACGCCCCCT GTCTTGGATA GCGACGGATC GTTTTTCCTC TACTCGAAAC TCACAGTAGA TAAGTCCCGA TGGCAACAGG GTAATGTCTT TAGCTGCAGC GTGATGCACG AGGCGCTTCA CAATCATTAC ACACAAAAAT CACTGTCGCT TAGCCCGGGA AAGGGTTCAG ATTCGTCGCC CCTGTTGCAG TTTGGTGGAC AGGTCAGACA GCGCTACCTT TACACGGATG ACGCCTGCCA GACAGAGGCA CACCTCGAAA TCAGAGAGGA CGGTACGGTC GGGGGTGCGG CCGATCAGAG CCCCGAGTCG CTTCTCCAGT TGAAGGCCCT TAAGCCAGGA GTCATCCAGA TTTTGGGAGT AAAGACCTCA CGGTTTCTCT GTCAGCGTCC AGATGGGACA CTGTACGGCT CATTGCATTT CGATCCCGAA GCGTGCTCGT TCCGGGAGTT GCTGCTTGAG GACGGATATA ACGTCTATCA GAGCGAAGCG CATGGCCTCC CCCTTCACCT CCCGTGTAAC AGGTCGCCGC ATCGGGATCC GGCCTCGAGG GGTCCCGCGA GATTTCTTCC GTTGCCCGGG TTGCCTCCCG CGCTGCCCGA GCCTCCCGGG ATCCTCGCGC CACAGCCTCC TGATGTAGGG TCCTCGGACC CTTTGGCGAT GGTAGGTGGA TCACAAGCAC GGTCCCCGAG CTATGCATCA 133 V210 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1- PSSGAPPPSD KTHTCPPCPA PEAAGGPSVF 39-Fc-GS- 39)-L0- LFPPKPKDTL MISRTPEVTC VVVDVSHEDP FGF21(33- Fc-L2- EVKFNWYVDG VEVHNAKTKP REEQYNSTYR 209; Q55C- FGF21 VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP R105K- (V103) IEKTISKAKG QPREPQVYTL PPSREEMTKN G148C- QVSLTCLVKG FYPSDIAVEW ESNGQPENNY K150R- KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN P158S- VFSCSVMHEA LHNHYTQKSL SLSPGKGSDS S195A- SPLLQFGGQV RQRYLYTDDA CQTEAHLEIR P199G- EDGTVGGAAD QSPESLLQLK ALKPGVIQIL G202A) GVKTSRFLCQ KPDGALYGSL HFDPEACSFR ELLLEDGYNV YQSEAHGLPL HLPCNRSPHR DPASRGPARF LPLPGLPPAL PEPPGILAPQ PPDVGSSDPL AMVGGSQARS PSYAS 56 CACGGAGAAG GCACCTTTAC ATCGGACTTG Exendin4(1- Ex(1- TCGAAGCAGA TGGAGGAAGA AGCGGTGAGG 39-Fc-GS- 39)-L0- CTCTTCATCG AGTGGCTCAA GAATGGAGGA FGF21(33- Fc-L2- CCCTCAAGCG GAGCGCCTCC TCCTTCCGAC 209; Q55C- FGF21 AAAACCCATA CATGTCCGCC TTGTCCCGCA R105K- (V103) CCAGAAGCAG CGGGTGGGCC CTCGGTGTTC G148C- CTGTTCCCGC CAAAACCGAA GGACACACTT K150R- ATGATTTCAC GCACACCGGA AGTGACTTGC P158S- GTCGTGGTGG ATGTATCGCA CGAGGACCCC S195A- GAGGTCAAAT TCAACTGGTA TGTCGATGGA P199G- GTGGAGGTGC ACAATGCAAA GACCAAGCCG G202A) AGGGAAGAAC AATACAATAG CACGTACCGA GTCGTGTCCG TCTTGACGGT CCTTCACCAG GACTGGCTGA ACGGAAAGGA GTACAAGTGC AAAGTGAGCA ATAAGGCCCT CCCTGCCCCG ATTGAGAAAA CCATTTCCAA GGCCAAAGGT CAGCCTAGAG AACCTCAAGT GTATACTCTT CCGCCCTCAC GCGAAGAGAT GACGAAAAAC CAAGTGTCGC TTACGTGTCT TGTCAAAGGT TTCTACCCCT CGGACATCGC CGTAGAGTGG GAGTCGAACG GCCAGCCGGA GAACAACTAC AAGACCACGC CCCCTGTCTT GGATAGCGAC GGATCGTTTT TCCTCTACTC GAAACTCACA GTAGATAAGT CCCGATGGCA ACAGGGTAAT GTCTTTAGCT GCAGCGTGAT GCACGAGGCG CTTCACAATC ATTACACACA AAAATCACTG TCGCTTAGCC CGGGAAAGGG TTCAGATTCG TCGCCCCTGT TGCAGTTTGG TGGACAGGTC AGACAGCGCT ACCTTTACAC GGATGACGCC TGCCAGACAG AGGCACACCT CGAAATCAGA GAGGACGGTA CGGTCGGGGG TGCGGCCGAT CAGAGCCCCG AGTCGCTTCT CCAGTTGAAG GCCCTTAAGC CAGGAGTCAT CCAGATTTTG GGAGTAAAGA CCTCACGGTT TCTCTGTCAG AAACCAGATG GGGCACTGTA CGGCTCATTG CATTTCGATC CCGAAGCGTG CTCGTTCCGG GAGTTGCTGC TTGAGGACGG ATATAACGTC TATCAGAGCG AAGCGCATGG CCTCCCCCTT CACCTCCCGT GTAACAGGTC GCCGCATCGG GATCCGGCCT CGAGGGGTCC CGCGAGATTT CTTCCGTTGC CCGGGTTGCC TCCCGCGCTG CCCGAGCCTC CCGGGATCCT CGCGCCACAG CCTCCTGATG TAGGGTCCTC GGACCCTTTG GCGATGGTAG GTGGATCACA AGCACGGTCC CCGAGCTATG CATCA 134 V211 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1- PSSGAPPPSG GGGSGGGGSG GGGSDKTHTC 39)- 39)- PPCPAPEAAG GPSVFLFPPK PKDTLMISRT (GGGGS)₃Fc- L15-Fc- PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN GS- L2- AKTKPREEQY NSTYRVVSVL TVLHQDWLNG FGF21(33- FGF21 KEYKCKVSNK ALPAPIEKTI SKAKGQPREP 209; Q55C- (V103) QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD R105K- IAVEWESNGQ PENNYKTTPP VLDSDGSFFL G148C- YSKLTVDKSR WQQGNVFSCS VMHEALHNHY K150R- TQKSLSLSPG KGSDSSPLLQ FGGQVRQRYL P158S- YTDDACQTEA HLEIREDGTV GGAADQSPES 5195A- LLQLKALKPG VIQILGVKTS RFLCQKPDGA P199G- LYGSLHFDPE ACSFRELLLE DGYNVYQSEA G202A) HGLPLHLPCN RSPHRDPASR GPARFLPLPG LPPALPEPPG ILAPQPPDVG SSDPLAMVGG SQARSPSYAS 57 CACGGAGAAG GCACCTTTAC ATCGGACTTG Exendin4(1- Ex(1- TCGAAGCAGA TGGAGGAAGA AGCGGTGAGG 39)- 39)- CTCTTCATCG AGTGGCTCAA GAATGGAGGA (GGGGS)₃Fc- L15-Fc- CCCTCAAGCG GAGCGCCTCC TCCTTCCGGA GS- L2- GGAGGTGGGT CGGGCGGTGG AGGCTCCGGA FGF21 FGF21 GGGGGAGGGA GCGACAAAAC CCATACATGT (33-209; Q55C- (V103) CCGCCTTGTC CCGCACCAGA AGCAGCGGGT R105K- GGGCCCTCGG TGTTCCTGTT CCCGCCAAAA G148C- CCGAAGGACA CACTTATGAT TTCACGCACA K150R- CCGGAAGTGA CTTGCGTCGT GGTGGATGTA P158S- TCGCACGAGG ACCCCGAGGT CAAATTCAAC 5195A- TGGTATGTCG ATGGAGTGGA GGTGCACAAT P199G- GCAAAGACCA AGCCGAGGGA AGAACAATAC G202A) AATAGCACGT ACCGAGTCGT GTCCGTCTTG ACGGTCCTTC ACCAGGACTG GCTGAACGGA AAGGAGTACA AGTGCAAAGT GAGCAATAAG GCCCTCCCTG CCCCGATTGA GAAAACCATT TCCAAGGCCA AAGGTCAGCC TAGAGAACCT CAAGTGTATA CTCTTCCGCC CTCACGCGAA GAGATGACGA AAAACCAAGT GTCGCTTACG  TGTCTTGTCA AAGGTTTCTA CCCCTCGGAC ATCGCCGTAG AGTGGGAGTC GAACGGCCAG CCGGAGAACA ACTACAAGAC CACGCCCCCT GTCTTGGATA GCGACGGATC GTTTTTCCTC TACTCGAAAC TCACAGTAGA TAAGTCCCGA TGGCAACAGG GTAATGTCTT TAGCTGCAGC GTGATGCACG AGGCGCTTCA CAATCATTAC ACACAAAAAT CACTGTCGCT TAGCCCGGGA AAGGGTTCAG ATTCGTCGCC CCTGTTGCAG TTTGGTGGAC AGGTCAGACA GCGCTACCTT TACACGGATG ACGCCTGCCA GACAGAGGCA CACCTCGAAA TCAGAGAGGA CGGTACGGTC GGGGGTGCGG CCGATCAGAG CCCCGAGTCG CTTCTCCAGT TGAAGGCCCT TAAGCCAGGA GTCATCCAGA TTTTGGGAGT AAAGACCTCA CGGTTTCTCT GTCAGAAACC AGATGGGGCA CTGTACGGCT CATTGCATTT CGATCCCGAA GCGTGCTCGT TCCGGGAGTT GCTGCTTGAG GACGGATATA ACGTCTATCA GAGCGAAGCG CATGGCCTCC CCCTTCACCT CCCGTGTAAC AGGTCGCCGC ATCGGGATCC GGCCTCGAGG GGTCCCGCGA GATTTCTTCC GTTGCCCGGG TTGCCTCCCG CGCTGCCCGA GCCTCCCGGG ATCCTCGCGC CACAGCCTCC TGATGTAGGG TCCTCGGACC CTTTGGCGAT GGTAGGTGGA TCACAAGCAC GGTCCCCGAG CTATGCATCA 135 V214 HSEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7- GLP- GGGSGGGGSG GGGSDKTHTC PPCPAPEAAG 35; A8S)- 1(A8S)- GPSVFLFPPK PKDTLMISRT PEVTCVVVDV (GGGGS)₃- L15-Fc- SHEDPEVKFN WYVDGVEVHN AKTKPREEQY Fc-GS- L2- NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK FGF21(33- FGF21 ALPAPIEKTI SKAKGQPREP QVYTLPPSRE 209; Q55C- (V101) EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ A109T- PENNYKTTPP VLDSDGSFFL YSKLTVDKSR G148C- WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K150R- KGSDSSPLLQ FGGQVRQRYL YTDDACQTEA P158S- HLEIREDGTV GGAADQSPES LLQLKALKPG S195A- VIQILGVKTS RFLCQRPDGT LYGSLHFDPE P199G-G202A) ACSFRELLLE DGYNVYQSEA HGLPLHLPCN RSPHRDPASR GPARFLPLPG LPPALPEPPG ILAPQPPDVG SSDPLAMVGG SQARSPSYAS 64 CACTCCGAAG GAACATTCAC TTCCGATGTA GLP-1(7- GLP- AGCTCGTATT TGGAAGGGCA GGCGGCTAAG 35; A8S)- 1(A8S)- GAGTTTATCG CATGGTTGGT CAAAGGTGGT (GGGGS)₃- L15-Fc- GGAGGTGGGT CGGGCGGTGG AGGCTCCGGA Fc-GS- L2- GGGGGAGGGA GCGACAAAAC CCATACATGT FGF21(33- FGF21 CCGCCTTGTC CCGCACCAGA AGCAGCGGGT 209; Q55C- (V101) GGGCCCTCGG TGTTCCTGTT CCCGCCAAAA A109T- CCGAAGGACA CACTTATGAT TTCACGCACA G148C- CCGGAAGTGA CTTGCGTCGT GGTGGATGTA K150R- TCGCACGAGG ACCCCGAGGT CAAATTCAAC P158S- TGGTATGTCG ATGGAGTGGA GGTGCACAAT S195A- GCAAAGACCA AGCCGAGGGA AGAACAATAC P199G- AATAGCACGT ACCGAGTCGT GTCCGTCTTG G202A) ACGGTCCTTC ACCAGGACTG GCTGAACGGA AAGGAGTACA AGTGCAAAGT GAGCAATAAG GCCCTCCCTG CCCCGATTGA GAAAACCATT TCCAAGGCCA AAGGTCAGCC TAGAGAACCT CAAGTGTATA CTCTTCCGCC CTCACGCGAA GAGATGACGA AAAACCAAGT GTCGCTTACG TGTCTTGTCA AAGGTTTCTA CCCCTCGGAC ATCGCCGTAG AGTGGGAGTC GAACGGCCAG CCGGAGAACA ACTACAAGAC CACGCCCCCT GTCTTGGATA GCGACGGATC GTTTTTCCTC TACTCGAAAC TCACAGTAGA TAAGTCCCGA TGGCAACAGG GTAATGTCTT TAGCTGCAGC GTGATGCACG AGGCGCTTCA CAATCATTAC ACACAAAAAT CACTGTCGCT TAGCCCGGGA AAGGGTTCAG ATTCGTCGCC CCTGTTGCAG TTTGGTGGAC AGGTCAGACA GCGCTACCTT TACACGGATG ACGCCTGCCA GACAGAGGCA CACCTCGAAA TCAGAGAGGA CGGTACGGTC GGGGGTGCGG CCGATCAGAG CCCCGAGTCG CTTCTCCAGT TGAAGGCCCT TAAGCCAGGA GTCATCCAGA TTTTGGGAGT AAAGACCTCA CGGTTTCTCT GTCAGCGTCC AGATGGGACA CTGTACGGCT CATTGCATTT CGATCCCGAA GCGTGCTCGT TCCGGGAGTT GCTGCTTGAG GACGGATATA ACGTCTATCA GAGCGAAGCG CATGGCCTCC CCCTTCACCT CCCGTGTAAC AGGTCGCCGC ATCGGGATCC GGCCTCGAGG GGTCCCGCGA GATTTCTTCC GTTGCCCGGG TTGCCTCCCG CGCTGCCCGA GCCTCCCGGG ATCCTCGCGC CACAGCCTCC TGATGTAGGG TCCTCGGACC CTTTGGCGAT GGTAGGTGGA TCACAAGCAC GGTCCCCGAG CTATGCATCA 136 V216 HSEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7- GLP- GGGSGGGGSG GGGSDKTHTC PPCPAPEAAG 35; A8S)- 1(A8S)- GPSVFLFPPK PKDTLMISRT PEVTCVVVDV (GGGGS)₃- L15-Fc- SHEDPEVKFN WYVDGVEVHN AKTKPREEQY Fc-GS- L2- NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK FGF21(33- FGF21 ALPAPIEKTI SKAKGQPREP QVYTLPPSRE 209; Q55C- (V103) EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ R105K- PENNYKTTPP VLDSDGSFFL YSKLTVDKSR G148C- WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K150R- KGSDSSPLLQ FGGQVRQRYL YTDDACQTEA 158S- HLEIREDGTV GGAADQSPES LLQLKALKPG S195A- VIQILGVKTS RFLCQKPDGA LYGSLHFDPE P199G- ACSFRELLLE DGYNVYQSEA HGLPLHLPCN G202A) RSPHRDPASR GPARFLPLPG LPPALPEPPG ILAPQPPDVG SSDPLAMVGG SQARSPSYAS 65 CACTCCGAAG GAACATTCAC TTCCGATGTA GLP-1(7- GLP- AGCTCGTATT TGGAAGGGCA GGCGGCTAAG 35; A8S)- 1(A8S)- GAGTTTATCG CATGGTTGGT CAAAGGTGGT (GGGGS)₃- L15-Fc- GGAGGTGGGT CGGGCGGTGG AGGCTCCGGA Fc-GS- L2- GGGGGAGGGA GCGACAAAAC CCATACATGT FGF21(33- FGF21 CCGCCTTGTC CCGCACCAGA AGCAGCGGGT 209; Q55C- (V103) GGGCCCTCGG TGTTCCTGTT CCCGCCAAAA R105K- CCGAAGGACA CACTTATGAT TTCACGCACA G148C- CCGGAAGTGA CTTGCGTCGT GGTGGATGTA K150R- TCGCACGAGG ACCCCGAGGT CAAATTCAAC P158S- TGGTATGTCG ATGGAGTGGA GGTGCACAAT S195A- GCAAAGACCA AGCCGAGGGA AGAACAATAC P199G- AATAGCACGT ACCGAGTCGT GTCCGTCTTG G202A) ACGGTCCTTC ACCAGGACTG GCTGAACGGA AAGGAGTACA AGTGCAAAGT GAGCAATAAG GCCCTCCCTG CCCCGATTGA GAAAACCATT TCCAAGGCCA AAGGTCAGCC TAGAGAACCT CAAGTGTATA CTCTTCCGCC CTCACGCGAA GAGATGACGA AAAACCAAGT GTCGCTTACG TGTCTTGTCA AAGGTTTCTA CCCCTCGGAC ATCGCCGTAG AGTGGGAGTC GAACGGCCAG CCGGAGAACA ACTACAAGAC CACGCCCCCT GTCTTGGATA GCGACGGATC GTTTTTCCTC TACTCGAAAC TCACAGTAGA TAAGTCCCGA TGGCAACAGG GTAATGTCTT TAGCTGCAGC GTGATGCACG AGGCGCTTCA CAATCATTAC ACACAAAAAT CACTGTCGCT TAGCCCGGGA AAGGGTTCAG ATTCGTCGCC CCTGTTGCAG TTTGGTGGAC AGGTCAGACA GCGCTACCTT TACACGGATG ACGCCTGCCA GACAGAGGCA CACCTCGAAA TCAGAGAGGA CGGTACGGTC GGGGGTGCGG CCGATCAGAG CCCCGAGTCG CTTCTCCAGT TGAAGGCCCT TAAGCCAGGA GTCATCCAGA TTTTGGGAGT AAAGACCTCA CGGTTTCTCT GTCAGAAACC AGATGGGGCA CTGTACGGCT CATTGCATTT CGATCCCGAA GCGTGCTCGT TCCGGGAGTT GCTGCTTGAG GACGGATATA ACGTCTATCA GAGCGAAGCG CATGGCCTCC CCCTTCACCT CCCGTGTAAC AGGTCGCCGC ATCGGGATCC GGCCTCGAGG GGTCCCGCGA GATTTCTTCC GTTGCCCGGG TTGCCTCCCG CGCTGCCCGA GCCTCCCGGG ATCCTCGCGC CACAGCCTCC TGATGTAGGG TCCTCGGACC CTTTGGCGAT GGTAGGTGGA TCACAAGCAC GGTCCCCGAG CTATGCATCA 137 V218 HSEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7- GLP- GGGSGGGGSG GGGSDKTHTC PPCPAPEAAG 35; A8S)- 1(A8S)- GPSVFLFPPK PKDTLMISRT PEVTCVVVDV (GGGGS)₃-Fc- L15-Fc- SHEDPEVKFN WYVDGVEVHN AKTKPREEQY FGF21(33- L0- NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK 209; Q55C- FGF21 ALPAPIEKTI SKAKGQPREP QVYTLPPSRE R105K- (V103) EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ G148C- PENNYKTTPP VLDSDGSFFL YSKLTVDKSR K150R- WQQGNVFSCS VMHEALHNHY TQKSLSLSPG P158S- KDSSPLLQFG GQVRQRYLYT DDACQTEAHL S195A- EIREDGTVGG AADQSPESLL QLKALKPGVI P199G- QILGVKTSRF LCQKPDGALY GSLHFDPEAC G202A) SFRELLLEDG YNVYQSEAHG LPLHLPCNRS PHRDPASRGP ARFLPLPGLP PALPEPPGIL APQPPDVGSS DPLAMVGGSQ ARSPSYAS 106 CACTCCGAAG GAACATTCAC TTCCGATGTA GLP-1(7- GLP-1(A8S)- AGCTCGTATT TGGAAGGGCA GGCGGCTAAG 35; A8S)- L15-Fc-L0- GAGTTTATCG CATGGTTGGT CAAAGGTGGT (GGGGS)₃-Fc- FGF21(V GGAGGTGGGT CGGGCGGTGG AGGCTCCGGA FGF21(33- 103) GGGGGAGGGA GCGACAAAAC CCATACATGT 209; Q55C- CCGCCTTGTC CCGCACCAGA AGCAGCGGGT R105K- GGGCCCTCGG TGTTCCTGTT CCCGCCAAAA G148C- CCGAAGGACA CACTTATGAT TTCACGCACA K150R- CCGGAAGTGA CTTGCGTCGT GGTGGATGTA P158S- TCGCACGAGG ACCCCGAGGT CAAATTCAAC S195A- TGGTATGTCG ATGGAGTGGA GGTGCACAAT P199G- GCAAAGACCA AGCCGAGGGA AGAACAATAC G202A) AATAGCACGT ACCGAGTCGT GTCCGTCTTG ACGGTCCTTC ACCAGGACTG GCTGAACGGA AAGGAGTACA AGTGCAAAGT GAGCAATAAG GCCCTCCCTG CCCCGATTGA GAAAACCATT TCCAAGGCCA AAGGTCAGCC TAGAGAACCT CAAGTGTATA CTCTTCCGCC CTCACGCGAA GAGATGACGA AAAACCAAGT GTCGCTTACG TGTCTTGTCA AAGGTTTCTA CCCCTCGGAC ATCGCCGTAG AGTGGGAGTC GAACGGCCAG CCGGAGAACA ACTACAAGAC CACGCCCCCT GTCTTGGATA GCGACGGATC GTTTTTCCTC TACTCGAAAC TCACAGTAGA TAAGTCCCGA TGGCAACAGG GTAATGTCTT TAGCTGCAGC GTGATGCACG AGGCGCTTCA CAATCATTAC ACACAAAAAT CACTGTCGCT TAGCCCGGGA AAGGATTCGT CGCCCCTGTT GCAGTTTGGT GGACAGGTCA GACAGCGCTA CCTTTACACG GATGACGCCT GCCAGACAGA GGCACACCTC GAAATCAGAG AGGACGGTAC GGTCGGGGGT GCGGCCGATC AGAGCCCCGA GTCGCTTCTC CAGTTGAAGG CCCTTAAGCC AGGAGTCATC CAGATTTTGG GAGTAAAGAC CTCACGGTTT CTCTGTCAGA AACCAGATGG GGCACTGTAC GGCTCATTGC ATTTCGATCC CGAAGCGTGC TCGTTCCGGG AGTTGCTGCT TGAGGACGGA TATAACGTCT ATCAGAGCGA AGCGCATGGC CTCCCCCTTC ACCTCCCGTG TAACAGGTCG CCGCATCGGG ATCCGGCCTC GAGGGGTCCC GCGAGATTTC TTCCGTTGCC CGGGTTGCCT CCCGCGCTGC CCGAGCCTCC CGGGATCCTC GCGCCACAGC CTCCTGATGT AGGGTCCTCG GACCCTTTG CGATGGTAGG TGGATCACAA GCACGGTCCC CGAGCTATGC ATCA 107 V200 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1- PSSGAPPPSG GGGSGGGGSG GGGSDKTHTC 39)- 39)-L15-Fc PPCPAPEAAG GPSVFLFPPK PKDTLMISRT (GGGGS)₃-Fc PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K 139 V201 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1- PSSGAPPPSG GGGSDKTHTC PPCPAPEAAG 39)-GGGGS- 39)-L5- GPSVFLFPPK PKDTLMISRT PEVTCVVVDV Fc Fc SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K 140 V207 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1- PSSGAPPPSD KTHTCPPCPA PEAAGGPSVF 39)-Fc- 39)-L0- LFPPKPKDTL MISRTPEVTC VVVDVSHEDP (GGGGS)₃- Fc-L15- EVKFNWYVDG VEVHNAKTKP REEQYNSTYR FGF21(33- FGF21 VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP 209; Q55C- (V103) IEKTISKAKG QPREPQVYTL PPSREEMTKN R105K- QVSLTCLVKG FYPSDIAVEW ESNGQPENNY G148C- KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN K150R- VFSCSVMHEA LHNHYTQKSL SLSPGKGGGG P158S- SGGGGSGGGG SDSSPLLQFG GQVRQRYLYT S195A- DDACQTEAHL EIREDGTVGG AADQSPESLL P199G- QLKALKPGVI QILGVKTSRF LCQKPDGALY G202A) GSLHFDPEAC SFRELLLEDG YNVYQSEAHG LPLHLPCNRS PHRDPASRGP ARFLPLPGLP PALPEPPGIL APQPPDVGSS DPLAMVGGSQ ARSPSYAS 141 V212 HSEGTFTSDV SSYLEGQAAK EFIAWLVKDK GLP-1(7- GLP- THTCPPCPAP EAAGGPSVFL FPPKPKDTLM 35; A8S)-Fc- 1(A8S)- ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV (GGGGS)₃- L0-Fc- EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD FGF21(33- L15- WLNGKEYKCK VSNKALPAPI EKTISKAKGQ 209; Q55C- FGF21 PREPQVYTLP PSREEMTKNQ VSLTCLVKGF R105K- (V103) YPSDIAVEWE SNGQPENNYK TTPPVLDSDG G148C- SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL K150R- HNHYTQKSLS LSPGKGGGGS GGGGSGGGGS P158S- DSSPLLQFGG QVRQRYLYTD DACQTEAHLE S195A- IREDGTVGGA ADQSPESLLQ LKALKPGVIQ P199G- ILGVKTSRFL CQKPDGALYG SLHFDPEACS G202A) FRELLLEDGY NVYQSEAHGL PLHLPCNRSP HRDPASRGPA RFLPLPGLPP ALPEPPGILA PQPPDVGSSD PLAMVGGSQA RSPSYAS 142 V213 HSEGTFTSDV SSYLEGQAAK EFIAWLVKDK GLP-1(7- GLP- THTCPPCPAP EAAGGPSVFL FPPKPKDTLM 35; A8S)-Fc- 1(A8S)- ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV GS- L0-Fc- EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD FGF21(33- L2- WLNGKEYKCK VSNKALPAPI EKTISKAKGQ 209; Q55C- FGF21 PREPQVYTLP PSREEMTKNQ VSLTCLVKGF A109T- (V101) YPSDIAVEWE SNGQPENNYK TTPPVLDSDG G148C- SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL K150R- HNHYTQKSLS LSPGKGSDSS PLLQFGGQVR P158S- QRYLYTDDAC QTEAHLEIRE DGTVGGAADQ 5195A- SPESLLQLKA LKPGVIQILG VKTSRFLCQR P199G- PDGTLYGSLH FDPEACSFRE LLLEDGYNVY G202A) QSEAHGLPLH LPCNRSPHRD PASRGPARFL PLPGLPPALP EPPGILAPQP PDVGSSDPLA MVGGSQARSP SYAS 143 V215 HSEGTFTSDV SSYLEGQAAK EFIAWLVKDK GLP-1(7- GLP- THTCPPCPAP EAAGGPSVFL FPPKPKDTLM 35; A8S)-Fc- 1(A8S)- ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV GS- L0-Fc- EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD FGF21(33- L2- WLNGKEYKCK VSNKALPAPI EKTISKAKGQ 209; Q55C- FGF21 PREPQVYTLP PSREEMTKNQ VSLTCLVKGF R105K- (V103) YPSDIAVEWE SNGQPENNYK TTPPVLDSDG G148C- SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL K150R- HNHYTQKSLS LSPGKGSDSS PLLQFGGQVR P158S- QRYLYTDDAC QTEAHLEIRE DGTVGGAADQ S195A- SPESLLQLKA LKPGVIQILG VKTSRFLCQK P199G- PDGALYGSLH FDPEACSFRE LLLEDGYNVY G202A) QSEAHGLPLH LPCNRSPHRD PASRGPARFL PLPGLPPALP EPPGILAPQP PDVGSSDPLA MVGGSQARSP SYAS 144 V217 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1- PSSGAPPPSG GGGSGGGGSG GGGSTDTLLL 39)- 39)- WVLLLWVPGS TGHGEGTFTS DLSKQMEEEA (GGGGS)₃- L15- VRLFIEWLKN GGPSSGAPPP SGGGGSGGGG Exendin4(1- Ex(1- SGGGGSDKTH TCPPCPAPEA AGGPSVFLFP 39)- 39)- PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK (GGGGS)3-Fc- L15-Fc- FNWYVDGVEV HNAKTKPREE QYNSTYRVVS GS- L2- VLTVLHQDWL NGKEYKCKVS NKALPAPIEK FGF21(33- FGF21 TISKAKGQPR EPQVYTLPPS REEMTKNQVS 209; Q55C- (V103) LTCLVKGFYP SDIAVEWESN GQPENNYKTT R105K- PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS G148C- CSVMHEALHN HYTQKSLSLS PGKGSDSSPL K150R- LQFGGQVRQR YLYTDDACQT EAHLEIREDG P158S- TVGGAADQSP ESLLQLKALK PGVIQILGVK 5195A- TSRFLCQKPD GALYGSLHFD PEACSFRELL P199G- LEDGYNVYQS EAHGLPLHLP CNRSPHRDPA G202A) SRGPARFLPL PGLPPALPEP PGILAPQPPD VGSSDPLAMV GGSQARSPSY AS 145 V278 HSEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7- GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD 35; A8S)- 1(A8S)- AQETEAHLEI REDGTVGGAA HQSPESLLEL GSGGGGSGGG- L10- KALKPGVIQI LGVKTSRFLC QKPDGALYGS FGF21(33- FGF21 LHFDPEACSF RELLLEDGYN VYQSEAHGLP 209; Q56E- (V76) LHLPGNRSPH CDPAPQGPAR FLPLPGLPPA D74H-Q82E- Dimer- LPEPPGILAP QPPDVGSSDP LAMVGPSQGR R105K- 40KPEGb SPSYAS K150R- R154C-bis- maleimide dimer 40 kDa branched PEG-R159Q- 5195A) 146 V279 HSEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7- GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD 35; A8S)- 1(A8S)- AQETEAHLEI REDGTVGGAA HQSPESLLEL GSGGGGSGGG- L10- KALKPGVIQI LGVKTSRFLC QKPDGALYGS FGF21(33- FGF21 LHFDPEACSF RELLLEDGYN VYQSEAHGLP 209; Q56E- (V76) LHLPGNRSPH CDPAPQGPAR FLPLPGLPPA D74H-Q82E- Dimer- LPEPPGILAP QPPDVGSSDP LAMVGPSQGR R105K- 20KPEGb SPSYAS K150R- R154C-bis- maleimide dimer 20 kDa branched PEG-R159Q- S195A) 147 V280 HSEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7- GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD 35; A8S)- 1(A8S)- AQETEAHLEI REDGTVGGAA HQSPESLLEL GSGGGGSGGG- L10- KALKPGVIQI LGVKTSRFLC QKPDGALYGS FGF21(33- FGF21 LHFDPEACSF RELLLEDGYN VYQSEAHGLP 209; Q56E- (V76) LHLPGNRSPH CDPAPQGPAR FLPLPGLPPA D74H-Q82E- LPEPPGILAP QPPDVGSSDP LAMVGPSQGR R105K- SPSYAS K150R- R154C- R159Q- S195A) 148 V283 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1- SGGGGSGGGG SGGGGSGGGG DSSPLLQFGG 30)- 30 - QVRQRYLYTD DAQETEAHLE IREDGTVGGA S(GGGGS)₃GGG L20- AHQSPESLLE LKALKPGVIQ ILGVKTSRFL G-FGF21(33- FGF21 CQKPDGALYG SLHFDPEACS FRELLLEDGY 209; Q56E- (V76) NVYQSEAHGL PLHLPGNRSP HCDPAPQGPA D74H-Q82E- RFLPLPGLPP ALPEPPGILA PQPPDVGSSD R105K- PLAMVGPSQG RSPSYAS K150R- R154C- R159Q- S195A) 149 V284 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1- SGGGGSGGGG SGGGGSGGGG DSSPLLQFGG 30)- 30)- QVRQRYLYTD DAQETEAHLE IREDGTVGGA S(GGGGS)₃GGGG- L20- AHQSPESLLE LKALKPGVIQ ILGVKTSRFL FGF21(33- FGF21 CQKPDGALYG SLHFDPEACS FRELLLEDGY 195; Q56E- (V76;  NVYQSEAHGL PLHLPGNRSP HCDPAPQGPA D74H-Q82E- CA14)- RFLPLPGLPP ALPEPPGILA PQPPDVGSSD R105K- 40KPEGb PLS K150R-R154C-40 kDa branched PEG-R159Q) 150 V285 DLSKQMEEEA VRLFIEWLKN GGSGGGGSGG Exendin4(9- Ex(9- GGSGGGGSGG GGDSSPLLQF GGQVRQRYLY 30)- 30)- TDDAQETEAH LEIREDGTVG GAAHQSPESL S(GGGGS)₃GGGG- L20- LELKALKPGV IQILGVKTSR FLCQKPDGAL FGF21(33- FGF21 YGSLHFDPEA CSFRELLLED GYNVYQSEAH 209; Q56E- (V76)- GLPLHLPGNR SPHCDPAPQG PARFLPLPGL D74H-Q82E- 40KPEGb PPALPEPPGI LAPQPPDVGS SDPLAMVGPS R105K- QGRSPSYAS K150R- R154C-40 kDa branched PEG-R159Q- S195A) 151 V289 GGQVRQRYLY TDDAQETEAH LEIREDGTVG FGF21(42- FGF21(V GAAHQSPESL LELKALKPGV IQILGVKTSR 209; Q56E- 76; NΔ9) FLCQKPDGAL YGSLHFDPEA CSFRELLLED D74H-Q82E- 40KPEGb GYNVYQSEAH GLPLHLPGNR SPHCDPAPQG R105K- PARFLPLPGL PPALPEPPGI LAPQPPDVGS K150R- SDPLAMVGPS QGRSPSYAS R154C-40 kDa branched PEG-R159Q- S195A) 152 V290 HSEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7- GLP- SGGGGSGGGG QVRQRYLYTD DAQETEAHLE 35; A8S)- 1(A8S)- IREDGTVGGA AHQSPESLLE LKALKPGVIQ GSGGGGSGGG- L10- ILGVKTSRFL CQKPDGALYG SLHFDPEACS FGF21(42- FGF21 FRELLLEDGY NVYQSEAHGL PLHLPGNRSP 209; Q56E- (V76; NΔ9)- HCDPAPQGPA RFLPLPGLPP ALPEPPGILA D74H-Q82E- 40KPEGb PQPPDVGSSD PLAMVGPSQG RSPSYAS R105K- K150R- R154C-40 kDa branched PEG-R159Q- S195A) 153 V291 GVSTSEAKFE QDSAILWYGV EFAKLHTSGG GLP-1(7- GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD 35; A8S 1(A8S; AQETEAHLEI REDGTVGGAA HQSPESLLEL scramble)- scramble)- KALKPGVIQI LGVKTSRFLC QKPDGALYGS GSGGGGSGGG- L10- LHFDPEACSF RELLLEDGYN VYQSEAHGLP FGF21(33- FGF21 LHLPGNRSPH CDPAPQGPAR FLPLPGLPPA 209; Q56E- (V76)- LPEPPGILAP QPPDVGSSDP LAMVGPSQGR D74H-Q82E- 40KPEGb SPSYAS R105K- K150R- R154C-40 kDa branched PEG-R159Q- S195A) 154 V311 HSEGTFTSDV SSYLEGQAAK EFIAWLVKGG GLP-1(7- GLP- SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD 35; A8S)- 1(A8S)- AQETEAHLEI REDGTVGGAA HQSPESLLEL GSGGGGSGGG- L10- KALKPGVIQI LGVKTSRFLC QKPDGALYGS FGF21(33- FGF21 LHFDPEACSF RELLLEDGYN VYQSEAHGLP 209; Q56E- (V76- LHLPGNRSPH CDPAPQGPAR FLPLPGLPPA D74H-Q82E- P199G)- LPEPPGILAP QPPDVGSSDP LAMVGGSQGR R105K- 40KPEGb SPSYAS K150R- R154C-40 kDa branched PEG-R159Q- S195A- P199G) 155 CATTCTGAAG GCACTTTTAC TAGCGATGTT GLP-1(7- GLP- TCTAGCTACC TGGAAGGCCA GGCTGCGAAA 35; A8S)- 1(A8S)- GAATTCATCG CGTGGCTGGT TAAAGGCGGT GSGGGGSGGG- L10- TCTGGTGGTG GTGGTTCTGG CGGTGGCGAC FGF21(33- FGF21 TCGAGCCCGC TGCTGCAATT TGGCGGCCAG 209; Q56E- (V76- GTGCGTCAGC GTTATCTGTA TACCGATGAT D74H-Q82E- P199G)- GCGCAGGAAA CCGAAGCGCA TCTGGAAATT R105K- 40KPEGb CGTGAAGATG GCACCGTGGG CGGTGCGGCG K150R- CATCAGAGCC CGGAAAGCCT GCTGGAACTG R154C-40 AAAGCGCTGA AACCGGGCGT GATTCAGATT kDa CTGGGCGTGA AAACCAGCCG TTTTCTGTGC branched CAGAAACCGG ATGGCGCGCT GTATGGCAGC PEG-R159Q- CTGCATTTTG ATCCGGAAGC GTGCAGCTTT S195A- CGTGAACTGC TGCTGGAAGA TGGCTATAAC P199G) GTGTATCAGA GCGAAGCGCA TGGCCTGCCG CTGCATCTGC CGGGCAACCG TAGCCCGCAT TGCGATCCGG CACCGCAGGG TCCGGCGCGT TTTCTGCCGC TGCCGGGTCT GCCGCCGGCA CTGCCGGAAC CGCCGGGTAT TCTGGCCCCG CAGCCGCCGG ATGTTGGTAG CAGCGATCCG CTGGCGATGG TGGGTGGTAG CCAGGGTCGT AGCCCGAGCT ATGCGAGC 156 V312 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1- SGGGGSGGGG SGGGGSGGGG DSSPLLQFGG 30)- 30)- QVRQRYLYTD DAQETEAHLE IREDGTVGGA S(GGGGS)₃GGGG- L20- AHQSPESLLE LKALKPGVIQ ILGVKTSRFL FGF21(33- FGF21 CQKPDGALYG SLHFDPEACS FRELLLEDGY 209; Q56E- (V76- NVYQSEAHGL PLHLPGNRSP HCDPAPQGPA D74H-Q82E- P199G)- RFLPLPGLPP ALPEPPGILA PQPPDVGSSD R105K- 40KPEGb PLAMVGGSQG RSPSYAS K150R- R154C-40 kDa branched PEG-R159Q- S195A- P199G) 157 CATGGTGAGG GTACGTTTAC TTCTGATCTG Exendin4(1- Ex(1- TCTAAACAGA TGGAAGAAGA AGCTGTTCGC 30)- 30)- CTGTTCATTG AATGGCTGAA AAATGGTGGT S(GGGGS)₃ L20- TCTGGTGGTG GTGGTTCTGG CGGTGGCGGT GGGG- FGF21 TCTGGCGGCG GTGGTAGCGG TGGCGGCGGT FGF21(33- (V76- GACTCGAGCC CGCTGCTGCA GTTTGGCGGC 209; Q56E- P199G)- CAGGTGCGTC AGCGTTATCT GTATACCGAT D74H-Q82E- 40KPEGb GATGCGCAGG AAACCGAAGC GCATCTGGAA R105K- ATTCGTGAAG ATGGCACCGT GGGCGGTGCG K150R- GCGCATCAGA GCCCGGAAAG CCTGCTGGAA R154C-40 CTGAAAGCGC TGAAACCGGG CGTGATTCAG kDa ATTCTGGGCG TGAAAACCAG CCGTTTTCTG branched TGCCAGAAAC CGGATGGCGC GCTGTATGGC PEG-R159Q- AGCCTGCATT TTGATCCGGA AGCGTGCAGC S195A-P199G) TTTCGTGAAC TGCTGCTGGA AGATGGCTAT AACGTGTATC AGAGCGAAGC GCATGGCCTG CCGCTGCATC TGCCGGGCAA CCGTAGCCCG CATTGCGATC CGGCACCGCA GGGTCCGGCG CGTTTTCTGC CGCTGCCGGG TCTGCCGCCG GCACTGCCGG AACCGCCGGG TATTCTGGCC CCGCAGCCGC CGGATGTTGG TAGCAGCGAT CCGCTGGCGA TGGTGGGTGG TAGCCAGGGT CGTAGCCCGA GCTATGCGAG C 158 V313 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1- PSSGAPPPSG GGGSGGGGSG GGGSDKTHTC 39)- 39)- PPCPAPEAAG GPSVFLFPPK PKDTLMISRT (GGGGS)₃-Fc- L15-Fc- PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN GS-FGF21 L2- AKTKPREEQY NSTYRVVSVL TVLHQDWLNG (33-208;  FGF21 KEYKCKVSNK ALPAPIEKTI SKAKGQPREP Q55C-R105K- (V103;  QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD G148C- CA1) IAVEWESNGQ PENNYKTTPP VLDSDGSFFL K150R- YSKLTVDKSR WQQGNVFSCS VMHEALHNHY P158S- TQKSLSLSPG KGSDSSPLLQ FGGQVRQRYL P174L- YTDDACQTEA HLEIREDGTV GGAADQSPES S195A- LLQLKALKPG VIQILGVKTS RFLCQKPDGA P199G- LYGSLHFDPE ACSFRELLLE DGYNVYQSEA G202A) HGLPLHLPCN RSPHRDPASR GPARFLPLPG LPPALPEPPG ILAPQPPDVG SSDPLAMVGG SQARSPSYA 159 V314 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1- PSSGAPPPSG GGGSGGGGSG GGGSDKTHTC 39)- 39)- PPCPAPEAAG GPSVFLFPPK PKDTLMISRT (GGGGS)₃-Fc- L15-Fc- PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN GS-FGF21 L2- AKTKPREEQY NSTYRVVSVL TVLHQDWLNG (33-207;  FGF21 KEYKCKVSNK ALPAPIEKTI SKAKGQPREP Q55C-R105K- (V103;  QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD G148C- CA2) IAVEWESNGQ PENNYKTTPP VLDSDGSFFL K150R- YSKLTVDKSR WQQGNVFSCS VMHEALHNHY P158S- TQKSLSLSPG KGSDSSPLLQ FGGQVRQRYL P174L- YTDDACQTEA HLEIREDGTV GGAADQSPES S195A- LLQLKALKPG VIQILGVKTS RFLCQKPDGA P199G- LYGSLHFDPE ACSFRELLLE DGYNVYQSEA G202A) HGLPLHLPCN RSPHRDPASR GPARFLPLPG LPPALPEPPG ILAPQPPDVG SSDPLAMVGG SQARSPSY 160 V315 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1- PSSGAPPPSG GGGSGGGGSG GGGSDKTHTC 39)- 39)- PPCPAPEAAG GPSVFLFPPK PKDTLMISRT (GGGGS)₃-Fc- L15-Fc- PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN GS-FGF21 L2- AKTKPREEQY NSTYRVVSVL TVLHQDWLNG (33-206;  FGF21 KEYKCKVSNK ALPAPIEKTI SKAKGQPREP Q55C-R105K- (V103;  QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD G148C- CA3) IAVEWESNGQ PENNYKTTPP VLDSDGSFFL K150R- YSKLTVDKSR WQQGNVFSCS VMHEALHNHY P158S- TQKSLSLSPG KGSDSSPLLQ FGGQVRQRYL P174L- YTDDACQTEA HLEIREDGTV GGAADQSPES S195A- LLQLKALKPG VIQILGVKTS RFLCQKPDGA P199G- LYGSLHFDPE ACSFRELLLE DGYNVYQSEA G202A) HGLPLHLPCN RSPHRDPASR GPARFLPLPG LPPALPEPPG ILAPQPPDVG SSDPLAMVGG SQARSPS 161 V316 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1- PSSGAPPPSG GGGSGGGGSG GGGSDKTHTC 39)- 39)- PPCPAPEAAG GPSVFLFPPK PKDTLMISRT (GGGGS)₃-Fc- L15-Fc- PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN GS-FGF21 L2- AKTKPREEQY NSTYRVVSVL TVLHQDWLNG (33-209;  FGF21 KEYKCKVSNK ALPAPIEKTI SKAKGQPREP Q55C-R105K- (V103)+ QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD G148C- P210 IAVEWESNGQ PENNYKTTPP VLDSDGSFFL K150R- YSKLTVDKSR WQQGNVFSCS VMHEALHNHY P158S- TQKSLSLSPG KGSDSSPLLQ FGGQVRQRYL P174L- YTDDACQTEA HLEIREDGTV GGAADQSPES S195A- LLQLKALKPG VIQILGVKTS RFLCQKPDGA P199G- LYGSLHFDPE ACSFRELLLE DGYNVYQSEA G202A)-P HGLPLHLPCN RSPHRDPASR GPARFLPLPG LPPALPEPPG ILAPQPPDVG SSDPLAMVGG SQARSPSYAS P 162 V317 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Exendin4(1- Ex(1- PSSGAPPPSG GGGSGGGGSG GGGSDKTHTC 39)- 39)- PPCPAPEAAG GPSVFLFPPK PKDTLMISRT (GGGGS)₃-Fc- L15-Fc- PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN GS-FGF21 L2- AKTKPREEQY NSTYRVVSVL TVLHQDWLNG (33-209;  FGF21 KEYKCKVSNK ALPAPIEKTI SKAKGQPREP Q55C-R105K- (V103- QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD G148C- S209A)+ IAVEWESNGQ PENNYKTTPP VLDSDGSFFL K150R- P210 YSKLTVDKSR WQQGNVFSCS VMHEALHNHY P158S- TQKSLSLSPG KGSDSSPLLQ FGGQVRQRYL P174L- YTDDACQTEA HLEIREDGTV GGAADQSPES S195A- LLQLKALKPG VIQILGVKTS RFLCQKPDGA P199G- LYGSLHFDPE ACSFRELLLE DGYNVYQSEA G202A- HGLPLHLPCN RSPHRDPASR GPARFLPLPG 5209A)-p LPPALPEPPG ILAPQPPDVG SSDPLAMVGG SQARSPSYAA P 163 V225 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGGSG GLP-1(7- GLP-1- DSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIR 35)-SG- L2- EDGTVGGAADQSPESLLQLKALKPGVIQILGV FGF21(33- FGF21 KTSRFLCQRPDGALYGSLHFDPEACSFRELLL 209; R154C) (154C) EDGYNVYQSEAHGLPLHLPGNKSPHCDPAPRG PARFLPLPGLPPALPEPPGILAPQPPDVGSSD PLSMVGPSQGRSPSYAS 164 V226 HSEGTFTSDVSSYLEGQAAKEFIAWLVKGGSG GLP-1(7-35;  GLP- DSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIR A8S) -SG- 1(A8S)- EDGTVGGAADQSPESLLQLKALKPGVIQILGV FGF21(33- L2- KTSRFLCQRPDGALYGSLHFDPEACSFRELLL 209; R154C) FGF21 EDGYNVYQSEAHGLPLHLPGNKSPHCDPAPRG (154C) PARFLPLPGLPPALPEPPGILAPQPPDVGSSD PLSMVGPSQGRSPSYAS 165 V229 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGGSG GLP-1(7- GLP-1-L2- DSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIR 35)-SG- FGF21 EDGTVGGAADQSPESLLQLKALKPGVIQILGV FGF21(33- (154C)- KTSRFLCQRPDGALYGSLHFDPEACSFRELLL 209; R154C- 40KPEG1 EDGYNVYQSEAHGLPLHLPGNKSPHCDPAPRG 40 kDa PARFLPLPGLPPALPEPPGILAPQPPDVGSSD linear PEG) PLSMVGPSQGRSPSYAS 166 V230 HSEGTFTSDVSSYLEGQAAKEFIAWLVKGGSG GLP-1(7-35;  GLP- DSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIR A8S)-SG- 1(A8S)- EDGTVGGAADQSPESLLQLKALKPGVIQILGV FGF21(33- L2- KTSRFLCQRPDGALYGSLHFDPEACSFRELLL 209; R154C- FGF21 EDGYNVYQSEAHGLPLHLPGNKSPHCDPAPRG 40 kDa (154C)- PARFLPLPGLPPALPEPPGILAPQPPDVGSSD linear PEG) 40KPEG1 PLSMVGPSQGRSPSYAS 167 V231 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGGSG GLP-1(7- GLP-1- GGGSGGGDSSPLLQFGGQVRQRYLYTDDAQQT 35)- L8- EAHLEIREDGTVGGAADQSPESLLQLKALKPG SGGGGSGGG- FGF21 VIQILGVKTSRFLCQRPDGALYGSLHFDPEAC FGF21(33- (154C)- SFRELLLEDGYNVYQSEAHGLPLHLPGNKSPH 209; R154C- 40KPEG1 CDPAPRGPARFLPLPGLPPALPEPPGILAPQP 40 kDa PDVGSSDPLSMVGPSQGRSPSYAS linear PEG) 168 V232 HSEGTFTSDVSSYLEGQAAKEFIAWLVKGGSG GLP-1(7-35;  GLP- GGGSGGGDSSPLLQFGGQVRQRYLYTDDAQQT A8S)- 1(A8S)- EAHLEIREDGTVGGAADQSPESLLQLKALKPG SGGGGSGGG- L8- VIQILGVKTSRFLCQRPDGALYGSLHFDPEAC FGF21(33- FGF21(1 SFRELLLEDGYNVYQSEAHGLPLHLPGNKSPH 209; R154C- 54C)- CDPAPRGPARFLPLPGLPPALPEPPGILAPQP 40 kDa 40KPEG1 PDVGSSDPLSMVGPSQGRSPSYAS linear PEG) 169 V237 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGGSG GLP-1(7- GLP-1- DSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIR 35)-SG- L2- EDGTVGGAADQSPESLLQLKALKPGVIQILGV FGF21(33- FGF21 KTSRFLCQRPDGALYGSLHFDPEACSFRELLL 209; R154C- (154C)- EDGYNVYQSEAHGLPLHLPGNKSPHCDPAPRG 40 kDa 40KPEGb PARFLPLPGLPPALPEPPGILAPQPPDVGSSD branched PLSMVGPSQGRSPSYAS PEG) 170 V238 GDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEI G-FGF21(33- FGF21 REDGTVGGAADQSPESLLQLKALKPGVIQILG 209; R154C- (R154C)- VKTSRFLCQRPDGALYGSLHFDPEACSFRELL 40 kDa 40KPEGb LEDGYNVYQSEAHGLPLHLPGNKSPHCDPAPR branched GPARFLPLPGLPPALPEPPGILAPQPPDVGSS PEG) DPLSMVGPSQGRSPSYAS 171 V253 HSEGTFTSDVSSYLEGQAAKEFIAWLVKGGSG GLP-1(7-35;  GLP- GGGSGGGDSSPLLQFGGQVRQRYLYTDDAQQT A8S)- 1(A8S)- EAHLEIREDGTVGGAADQSPESLLQLKALKPG SGGGGSGGG- L8- VIQILGVKTSRFLCQRPDGALYGSLHFDPEAC FGF21(33- FGF21- SFRELLLEDGYNVYQSEAHGLPLHLPGNKSPH 194; R154C- (C414;  CDPAPRGPARFLPLPGLPPALPEPPGILAPQP 40 kDa 154C)- PDVGSSDPLS branched 40KPEGb PEG) 172 V258 HGEGTFTSDVSSYLEGQAAKEFIAWLVKGGSG GLP-1(7-35;  GLP- GGGSGGGDSSPLLQFGGQVRQRYLYTDDAQQT A8G)- 1(A8G)- EAHLEIREDGTVGGAADQSPESLLQLKALKPG SGGGGSGGG- L8- VIQILGVKTSRFLCQRPDGALYGSLHFDPEAC FGF21(33- FGF21 SFRELLLEDGYNVYQSEAHGLPLHLPGNKSPH 209; R154C- (154C)- CDPAPRGPARFLPLPGLPPALPEPPGILAPQP 40 kDa 40KPEGb PDVGSSDPLSMVGPSQGRSPSYAS branched PEG)

The variants or mutants used in the proteins of the invention, e.g., variants of wild-type FGF21, GLP-1, and/or Exendin-4 feature at least one substituted, added, and/or removed amino acid relative to the wild-type protein. Additionally, the variants may include N- and/or C-terminal truncations relative to the wild-type proteins. Generally speaking, a variant possesses some modified property, structural or functional, of the wild-type protein. For example, the variant may have enhanced or improved physical stability in concentrated solutions (e.g., less hydrophobic mediated aggregation), enhanced or improved plasma stability when incubated with blood plasma or reduced risk for immunogenicity or enhanced or improved bioactivity while maintaining a favorable bioactivity profile.

Acceptable amino acid substitutions and modifications which constitute differences between the portions of the proteins of the invention and their wild-type comparator proteins include, but are not limited to, one or more amino acid substitutions, including substitutions with pyrrolysine, pyrroline-carbyoxy-lysine (Pcl) and non-naturally occurring amino acid analogs, and truncations. Thus, the proteins of the invention (e.g., the fusion proteins of the invention) include, but are not limited to, site-directed mutants, truncated polypeptides, proteolysis-resistant mutants, aggregation-reducing mutants, combination mutants, and fusion proteins, as described herein.

One skilled in the art of expression of proteins will recognize that methionine or methionine-arginine sequence can be introduced at the N-terminus of any of the proteins of the invention, for expression in E. coli, and are contemplated within the context of this invention.

One skilled in the art of expression of proteins will recognize that additional tags or fusion domains for the purposes of modulating expression levels, purification, or stabilization can be introduced to the N-terminus of any of the proteins of the invention, with or without an additional peptide to target digestion by a specific protease for later removal of that tag or fusion domain, for expression in any host cell, and are contemplated within the context of this invention.

One skilled in the art of expression of proteins will recognize that leader peptides targeting the expressed protein to the periplasm or extracellular space can be introduced at the N-terminus of any of the proteins of the invention, for expression in E. coli or other bacterial hosts, and are contemplated within the context of this invention.

One skilled in the art of expression of proteins will recognize that leader peptides targeting the expressed protein to the ER, secretory vesicles, or extracellular space can be introduced at the N-terminus of any of the proteins of the invention, for expression in eukaryotic host cells, and are contemplated within the context of this invention.

The proteins of the invention may possess increased compatibility with pharmaceutical preservatives (e.g., m-cresol, phenol, benzyl alcohol), thus enabling the preparation of a preserved pharmaceutical formulation that maintains the physiochemical properties and biological activity of the protein during storage. Accordingly, variants with enhanced pharmaceutical stability relative to wild-type, have improved physical stability in concentrated solutions under both physiological and preserved pharmaceutical formulation conditions, while maintaining biological potency. By way of non-limiting example, the proteins of the invention may be more resistant to proteolysis and enzymatic degradation; may have improved stability; and may be less likely to aggregate, than their wild-type counterparts or corresponding native sequence. As used herein, these terms are not mutually exclusive or limiting, it being entirely possible that a given variant has one or more modified properties of the wild-type protein.

The invention also encompasses nucleic acid molecules encoding the proteins of the invention, comprising, for example, an FGF21 amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:3, but wherein specific residues conferring a desirable property to the FGF21 protein variant, e.g., improved potency to FGF21-receptors, proteolysis-resistance, increased half-life or aggregation-reducing properties and combinations thereof have not been further modified. In other words, with the exception of residues in the FGF21 mutant sequence that have been modified in order to confer proteolysis-resistance, aggregation-reducing, or other properties, about 5% (alternately 4%, alternately 3%, alternately 2%, alternately 1%) of all other amino acid residues in the FGF21 mutant sequence can be modified. Such FGF21 mutants possess at least one activity of the wild-type FGF21 polypeptide.

Similarly, the invention also comprises nucleic acid molecules encoding the GLP-1 and Exendin-4 portions of the molecule, whose amino acid sequences are at least about 85%, identical, and more preferably at least about 90 to 95% identical, to the amino acid sequence of SEQ ID NO:30 and 7, respectively, but wherein specific residues conferring a desirable property to the dual function protein variant, e.g., proteolysis-resistance, increased half-life or aggregation-reducing properties and combinations thereof have not been further modified.

The invention also encompasses a nucleic acid molecule comprising a nucleotide sequence that is at least about 85%, identical, and more preferably, at least about 90 to 95% identical to the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and the cDNA sequence encoding wild-type Exendin-4, but wherein the nucleotides encoding amino acid residues conferring the encoded protein's proteolysis-resistance, aggregation-reducing or other properties have not been further modified. In other words, with the exception of nucleotides that encode residues in the FGF21, GLP-1, or Exendin-4 mutant sequences that have been modified in order to confer proteolysis-resistance, aggregation-reducing, or other properties, about 15%, and more preferably about 10 to 5% of all other nucleotides in the mutant sequence can be modified. Such nucleic acid molecules encode proteins possessing at least one activity of their wild-type counterparts.

Provided herein are methods used to generate the proteins of the invention, wherein such methods involve site-specific modification and non-site-specific modification of the wild-type versions of the proteins (e.g., the FGF21 wild-type protein as described herein), e.g., truncations of the wild-type proteins, and the site-specific incorporation of amino acids at positions of interest within the wild-type proteins. Said modifications enhance the biological properties of the proteins of the invention relative to the wild-type proteins, as well as, in some cases, serving as points of attachment for, e.g., labels and protein half-life extension agents, and for purposes of affixing said variants to the surface of a solid support. Related embodiments of the invention are methods of producing cells capable of producing said dual function proteins of the invention, and of producing vectors containing DNA encoding said variants.

In certain embodiments, such modifications, e.g., site-specific modifications, are used to attach conjugates, e.g., PEG groups to proteins, polypeptides, and/or peptides of the invention, for purposes of, e.g., extending half-life or otherwise improving the biological properties of said proteins, polypeptides, and/or peptides. Said techniques are described further herein.

In other embodiments, such modifications, e.g., site-specific modifications are used to attach other polymers, small molecules and recombinant protein sequences that extend half-life of the protein of the invention. One such embodiment includes the attachment of fatty acids or specific albumin binding compounds to proteins, polypeptides, and/or peptides. In other embodiments, the modifications are made at a particular amino acid type and may be attached at one or more sites on the protein.

In other embodiments, such modifications, e.g., site-specific modifications are used as means of attachment for the production of wild-type and/or variant multimers, e.g., dimers (homodimers or heterodimers) or trimers or tetramers. These multimeric protein molecules may additionally have groups such as PEG, sugars, and/or PEG-cholesterol conjugates attached or be fused either amino-terminally or carboxy-terminally to other proteins such as Fc, Human Serum Albumin (HSA), etc.

In other embodiments, such site-specific modifications are used to produce proteins, polypeptides and/or peptides wherein the position of the site-specifically incorporated pyrrolysine, pyrroline-carboxy-lysine, or pyrrolysine analogue or non-naturally occurring amino acids (para-acetyl-Phe, para-azido-Phe) allows for controlled orientation and attachment of such proteins, polypeptides and/or peptides onto a surface of a solid support or to have groups such as PEG, sugars and/or PEG-cholesterol conjugates attached.

In other embodiments, such site-specific modifications are used to site-specifically cross-link proteins, polypeptides and/or peptides thereby forming hetero-oligomers including, but not limited to, heterodimers and heterotrimers. In other embodiments, such site-specific modifications are used to site-specifically cross-link proteins, polypeptides and/or peptides thereby forming protein-protein conjugates, protein-polypeptide conjugates, protein-peptide conjugates, polypeptide-polypeptide conjugates, polypeptide-peptide conjugates or peptide-peptide conjugates. In other embodiments, a site specific modification may include a branching point to allow more than one type of molecule to be attached at a single site of a protein, polypeptide or peptide.

In other embodiments, the modifications listed herein can be done in a non-site-specific manner and result in protein-protein conjugates, protein-polypeptide conjugates, protein-peptide conjugates, polypeptide-polypeptide conjugates, polypeptide-peptide conjugates or peptide-peptide conjugates of the invention.

Definitions

Various definitions are used throughout this document. Most words have the meaning that would be attributed to those words by one skilled in the art. Words specifically defined either below or elsewhere in this document have the meaning provided in the context of the present invention as a whole and as are typically understood by those skilled in the art.

As used herein, the term “FGF21” refers to a member of the fibroblast growth factor (FGF) protein family. An amino acid sequence of FGF21 (GenBank Accession No. NP_061986.1) is set forth as SEQ ID NO:1, the corresponding polynucleotide sequence of which is set forth as SEQ ID NO:2 (NCBI reference sequence number NM_019113.2). “FGF21 variant,” “FGF21 mutant,” and similar terms describe modified version of the FGF21 protein, e.g., with constituent amino acid residues deleted, added, modified, or substituted.

As used herein, the term “FGF21 receptor” refers to a receptor for FGF21 (Kharitonenkov, A, et al. (2008) Journal of Cellular Physiology 215:1-7; Kurosu, H et al. (2007) JBC 282:26687-26695; Ogawa, Y et al. (2007) PNAS 104:7432-7437). The term “FGF21 polypeptide” refers to a naturally-occurring polypeptide expressed in humans. For purposes of this disclosure, the term “FGF21 polypeptide” can be used interchangeably to refer to any full-length FGF21 polypeptide, e.g., SEQ ID NO:1, which consists of 209 amino acid residues and which is encoded by the nucleotide sequence of SEQ ID NO:2; any mature form of the polypeptide, which consists of 181 amino acid residues, and in which the 28 amino acid residues at the amino-terminal end of the full-length FGF21 polypeptide (i.e., which constitute the signal peptide) have been removed.

“Variant 76,” or “V76,” as used herein, is an FGF21 protein variant, featuring a 40 kDa branched PEG linked through Cys154, and eight point mutations relative to the 177 amino acid wild-type protein, for example, SEQ ID NO: 185 or SEQ ID NO: 186 which is v76 with a P199G mutation. Synthesis of the variant is described in greater detail herein.

“Variant 101,” or “V101,” as used herein, is an FGF21 protein variant, featuring an engineered disulfide bridge, and eight point mutations relative to the 177 amino acid wild-type protein, for example, SEQ ID NO: 187, expressed as a fusion to human IgG1 Fc-domain with a GS linker. Synthesis of the variant is described in greater detail herein.

“Variant 103,” or “V103,” as used herein, is an FGF21 protein variant, featuring an engineered disulfide bridge, and eight point mutations relative to the 177 amino acid wild-type protein, for example, SEQ ID NO: 188, expressed as a fusion to human IgG1 Fc-domain with a GS linker. Synthesis of the variant is described in greater detail herein.

“Variant 188,” or “V188,” as used herein, is an FGF21 protein variant, featuring an engineered disulfide bridge, and eight point mutations relative to the 177 amino acid wild-type protein, expressed as a fusion to human IgG1 Fc-domain with a (SGGGG)₃ linker. Synthesis of the variant is described in greater detail herein

“GLP-1-FGF21-PEG dual agonists,” “dual function proteins,” “dual function fusion proteins,” “dual activity proteins,” “fusion products,” “dual FGF21 receptor agonist and GLP-1 receptor agonist,” “dual FGF21 receptor agonist and GLP-1 receptor agonist proteins of the invention,” “GLP-1-FGF21 fusion proteins,” “fusion proteins of the invention,” “fusions of the invention,” and similar terms define protein or polypeptide fusions comprising at least an FGF21 polypeptide or protein variant, mutant, or truncated version, fused or linked to another metabolic regulator such as GLP-1 or Exendin-4. They comprise a single molecule with dual activity or dual function vis-à-vis the receptors of their respective constituents, i.e., they show the ability to agonize the FGF21 receptor and the GLP-1 receptor. The constituent sequences of said fusion proteins may comprise modifications, truncations, other variants of naturally occurring (i.e., wild-type) protein or polypeptide counterparts, and may employ any number of various other modifications, e.g., PEG groups for half-life extension.

A particularly preferred embodiment of the GLP-1-FGF21-PEG fusion protein of the invention incorporates V76 (as defined herein) as the FGF21 variant. Said preferred embodiment is also referred to herein as “GLP-1(A8S)-FGF21-PEG” and features a substitution from alanine to serine at position 8 relative to the wild-type GLP-1 sequence (SEQ ID NO:5129) and substitution from arginine to cysteine at position 154 relative to the wild-type FGF21 sequence (SEQ ID:1). The sequence of GLP-1(A8S)-FGF21(V76)-PEG is as follows (SEQ ID NO:9).

  1 HSEGTFTSDV SSYLEGQAAK EFIAWLVKGG SGGGGSGGGD SSPLLQFGGQ VRQRYLYTDD  61 AQETEAHLEI REDGTVGGAA HQSPESLLEL KALKPGVIQI LGVKTSRFLC QKPDGALYGS 121 LHFDPEACSF RELLLEDGYN VYQSEAHGLP LHLPGNRSPH CDPAPQGPAR FLPLPGLPPA 181 LPEPPGILAP QPPDVGSSDP LAMVGPSQGR SPSYAS

The term “isolated nucleic acid molecule” refers to a nucleic acid molecule of the present invention that (1) has been separated from at least about 50 percent of proteins, lipids, carbohydrates, or other materials with which it is naturally found when total nucleic acid is isolated from the source cells, (2) is not linked to all or a portion of a polynucleotide to which the “isolated nucleic acid molecule” is linked in nature, (3) is operably linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature as part of a larger polynucleotide sequence. Preferably, the isolated nucleic acid molecule of the present invention is substantially free from any other contaminating nucleic acid molecules or other contaminants that are found in its natural environment that would interfere with its use in polypeptide production or its therapeutic, diagnostic, prophylactic or research use.

The term “vector” is used to refer to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information to a host cell.

The term “expression vector” refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control the expression of inserted heterologous nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing, if introns are present.

The term “operably linked” is used herein to refer to an arrangement of flanking sequences wherein the flanking sequences so described are configured or assembled so as to perform their usual function. Elements of fusions proteins may be operably linked to one another so as to allow the fusion protein to function as if it were a naturally occurring, endogenous protein, and/or to combine disparate elements of said fusion proteins in a synergistic fashion.

On a nucleotide level, a flanking sequence operably linked to a coding sequence may be capable of effecting the replication, transcription and/or translation of the coding sequence. For example, a coding sequence is operably linked to a promoter when the promoter is capable of directing transcription of that coding sequence. A flanking sequence need not be contiguous with the coding sequence, so long as it functions correctly. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.

The term “host cell” is used to refer to a cell which has been transformed, or is capable of being transformed with a nucleic acid sequence and then of expressing a selected gene of interest. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent, so long as the selected gene is present.

The term “amino acid,” as used herein, refers to naturally occurring amino acids, unnatural amino acids, amino acid analogues and amino acid mimetics that function in a manner similar to the naturally occurring amino acids, all in their D and L stereoisomers if their structure allows such stereoisomeric forms. Amino acids are referred to herein by either their name, their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature

Commission.

The term “naturally occurring” when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refers to materials which are found in nature and are not manipulated by man. Similarly, “non-naturally occurring” as used herein refers to a material that is not found in nature or that has been structurally modified or synthesized by man. When used in connection with nucleotides, the term “naturally occurring” refers to the bases adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U). When used in connection with amino acids, the term “naturally occurring” refers to the 20 conventional amino acids (i.e., alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y)), as well as selenocysteine, pyrrolysine (Pyl, or O), and pyrroline-carboxy-lysine (Pcl, or Z).

Pyrrolysine (Pyl) is an amino acid naturally found within methylamine methyltransferases of methanogenic archaea of the family Methanosarcina. Pyrrolysine is a lysine analogue co-translationally incorporated at in-frame UAG codons in the respective mRNA, and it is considered the 22nd natural amino acid.

As described at least in PCT patent publication WO2010/48582 (applicant IRM, LLC), attempts to biosynthesize pyrrolysine (Pyl) in E. coli resulted in the formation of a “demethylated pyrrolysine,” referred to herein as pyrroline-carboxy-lysine, or Pcl. “Pcl,” as used herein, refers to either Pcl-A or Pcl-B.

The terms “non-natural amino acid” and “unnatural amino acid,” as used herein, are interchangeably intended to represent amino acid structures that cannot be generated biosynthetically in any organism using unmodified or modified genes from any organism, whether the same or different. The terms refer to an amino acid residue that is not present in the naturally occurring (wild-type) FGF21 protein sequence or the sequences of the present invention. These include, but are not limited to, modified amino acids and/or amino acid analogues that are not one of the 20 naturally occurring amino acids, selenocysteine, pyrrolysine (Pyl), or pyrroline-carboxy-lysine (Pcl, e.g., as described in PCT patent publication WO2010/48582). Such non-natural amino acid residues can be introduced by substitution of naturally occurring amino acids, and/or by insertion of non-natural amino acids into the naturally occurring (wild-type) FGF21 protein sequence or the sequences of the invention. The non-natural amino acid residue also can be incorporated such that a desired functionality is imparted to the FGF21 molecule, for example, the ability to link a functional moiety (e.g., PEG). When used in connection with amino acids, the symbol “U” shall mean “non-natural amino acid” and “unnatural amino acid,” as used herein.

In addition, it is understood that such “unnatural amino acids” typically require a modified tRNA and a modified tRNA synthetase (RS) for incorporation into a protein. These “selected” orthogonal tRNA/RS pairs are generated by a selection process as developed by Schultz et al. or by random or targeted mutation. As way of example, pyrroline-carboxy-lysine is a “natural amino acid” as it is generated biosynthetically by genes transferred from one organism into the host cells and as it is incorporated into proteins by using natural tRNA and tRNA synthetase genes, while p-aminophenylalanine (See, Generation of a bacterium with a 21 amino acid genetic code, Mehl R A, Anderson J C, Santoro S W, Wang L, Martin A B, King D S, Horn D M, Schultz P G. J Am Chem Soc. 2003 Jan. 29; 125(4):935-9) is an “unnatural amino acid” because, although generated biosynthetically, it is incorporated into proteins by a “selected” (i.e. not “naturally-occurring”) orthogonal tRNA/tRNA synthetase pair.

Modified encoded amino acids include, but are not limited to, hydroxyproline, γ-carboxyglutamate, 0-phosphoserine, azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tertiary-butylglycine, 2,4-diaminoisobutyric acid, desmosine, 2,2′-diaminopimelic acid, 2,3-diaminoproprionic acid, N-ethylglycine, N-methylglycine, N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline, norleucine, ornithine, pentylglycine, pipecolic acid and thioproline. The term “amino acid” also includes naturally occurring amino acids that are metabolites in certain organisms but are not encoded by the genetic code for incorporation into proteins. Such amino acids include, but are not limited to, ornithine, D-ornithine, and D-arginine.

The term “amino acid analogue,” as used herein, refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, by way of example only, an α-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group. Amino acid analogues include the natural and unnatural amino acids which are chemically blocked, reversibly or irreversibly, or their C-terminal carboxy group, their N-terminal amino group and/or their side-chain functional groups are chemically modified. Such analogues include, but are not limited to, methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine sulfoxide, S-(carboxymethyl)-cysteine sulfone, aspartic acid-(beta-methyl ester), N-ethylglycine, alanine carboxamide, homoserine, norleucine, and methionine methyl sulfonium.

The term “amino acid mimetics,” as used herein, refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but functions in a manner similar to a naturally occurring amino acid.

The term “biologically active variant” refers to any polypeptide variant used in the dual function proteins of the invention, e.g., as a constituent protein of the fusions, that possesses an activity of its wild-type (e.g., naturally-occurring) protein or polypeptide counterpart, such as the ability to modulate blood glucose, HbAlc,□ insulin, triglyceride, or cholesterol levels; increase pancreatic function; reduce lipid levels in liver; reduce body weight; and to improve glucose tolerance, energy expenditure, or insulin sensitivity, regardless of the type or number of modifications that have been introduced into the polypeptide variant. Polypeptide variants possessing a somewhat decreased level of activity relative to their wild-type versions can nonetheless be considered to be biologically active polypeptide variants. A non-limiting representative example of a biologically active polypeptide variant of the invention is an FGF21 variant, which is modified after, and possesses similar or enhanced biological properties relative to, wild-type FGF21.

The terms “effective amount” and “therapeutically effective amount” each refer to the amount of a fusion protein of the invention used to support an observable level of one or more biological activities of the wild-type polypeptide or protein counterparts, such as the ability to lower blood glucose, insulin, triglyceride or cholesterol levels; reduce liver triglyceride or lipid levels; reduce body weight; or improve glucose tolerance, energy expenditure, or insulin sensitivity. For example, a “therapeutically-effective amount” administered to a patient exhibiting, suffering, or prone to suffer from FGF21-associated disorders or GLP-1-associated disorders (such as type 1 or type 2 diabetes mellitus, obesity, or metabolic syndrome), is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression, physiological conditions associated with or resistance to succumbing to the afore mentioned disorders. For the purposes of the present invention a “subject” or “patient” is preferably a human, but can also be an animal, more specifically, a companion animal (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, and the like).

The term “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” as used herein refers to one or more formulation materials suitable for accomplishing or enhancing the delivery of a fusion protein of the invention.

The term “antigen” refers to a molecule or a portion of a molecule that is capable of being bound by an antibody, and additionally that is capable of being used in an animal to produce antibodies that are capable of binding to an epitope of that antigen. An antigen may have one or more epitopes.

The term “native Fc” refers to molecule or sequence comprising the sequence of a non-antigen-binding fragment resulting from digestion of whole antibody or produced by other means, whether in monomeric or multimeric form, and can contain the hinge region. The original immunoglobulin source of the native Fc is preferably of human origin and can be any of the immunoglobulins, although IgG1 and IgG2 are preferred. Native Fc molecules are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG (see Ellison et al., 1982, Nucleic Acids Res. 10: 4071-9). The term “native Fc” as used herein is generic to the monomeric, dimeric, and multimeric forms. The term “Fc variant” refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn (neonatal Fc receptor). International Publication Nos. WO 97/34631 and WO 96/32478 describe exemplary Fc variants, as well as interaction with the salvage receptor, and are hereby incorporated by reference. Thus, the term “Fc variant” can comprise a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises regions that can be removed because they provide structural features or biological activity that are not required for the fusion molecules of the Proteins of the invention. Thus, the term “Fc variant” comprises a molecule or sequence that lacks one or more native Fc sites or residues, or in which one or more Fc sites or residues has be modified, that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC). Fc variants are described in further detail hereinafter.

The term “Fc domain” encompasses native Fc and Fc variants and sequences as defined above. As with Fc variants and native Fc molecules, the term “Fc domain” includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means. In some embodiments of the present invention, an Fc domain can be fused to FGF21 or a FGF21 mutant (including a truncated form of FGF21 or a FGF21 mutant) via, for example, a covalent bond between the Fc domain and the FGF21 sequence. Such fusion proteins can form multimers via the association of the Fc domains and both these fusion proteins and their multimers are an aspect of the present invention.

As used in the present text, the term “fusobody” refers to an antibody-like soluble protein comprising two heterodimers, each heterodimer consisting of one heavy and one light chains of amino acids, stably associated together, for example, via one or more disulfide bond(s). Each heavy or light chain comprises constant regions of an antibody, referred hereafter respectively as the heavy and light chain constant regions of the fusobody. The heavy chain constant region comprises at least C_(H)1 region of an antibody and may further comprise C_(H)2 and C_(H)3 regions, including the hinge region. The light chain constant region comprises C_(L) region of an antibody. In a fusobody, the variable regions of an antibody are replaced by heterologous soluble binding domains. By way of non-limiting example, a fusobody of the invention can comprise a dual function fusion protein of the invention, wherein the GLP-1 receptor agonist is fused to the N-terminus of heavy and light chain of an antibody and FGF21 is simultaneously fused to the C-terminus of heavy and light chain of the same antibody.

The term “heterologous” means that these domains are not naturally found associated with constant regions of an antibody. In particular, such heterologous binding domains do not have the typical structure of an antibody variable domain consisting of 4 framework regions, FR1, FR2, FR3 and FR4 and the 3 complementarity determining regions (CDRs) in-between. Each arm of the fusobody therefore comprises a first single chain polypeptides comprising a first binding domain covalently linked at the N-terminal part of a constant C_(H)1 heavy chain region of an antibody, and a second single chain polypeptide comprising a second binding domain covalently linked at the N-terminal part of a constant C_(L) light chain of an antibody. The covalent linkage may be direct, for example via peptidic bound or indirect, via a linker, for example a peptidic linker. The two heterodimers of the fusobody are covalently linked, for example, by at least one disulfide bridge at their hinge region, like an antibody structure. Examples of molecules with a fusobody structure have been described in the art, in particular, fusobodies comprising ligand binding region of heterodimeric receptor (see for example international patent publications WO01/46261 and WO11/076781).

The term “polyethylene glycol” or “PEG” refers to a polyalkylene glycol compound or a derivative thereof, with or without coupling agents or derviatization with coupling or activating moieties.

The term “Exenatide” indicates a synthetic version of exendin-4, Exenatide, marketed as Byetta and Bydureon, is a glucagon-like peptide-1 agonist (GLP-1 agonist) medication approved in April 2005 for the treatment of diabetes mellitus type 2. It belongs to the group of incretin mimetics and is manufactured by Amylin Pharmaceuticals.

Exendin-4 is a 39 residue polypeptide produced in the salivary glands of the Gila Monster lizard (Goke et al. (1993) Diabetes 46:433-439; Fehmann et al. (1995) Endocrine Rev. 16:390-410). Although it is the product of a uniquely non-mammalian gene and appears to be expressed only in the salivary gland, Exendin-4 shares a 52% amino acid sequence homology with GLP-1 and in mammals interacts with the GLP-1 receptor (Goke, et al.; Thorens et al. (1993) Diabetes 42:1678-1682).

The term “FGF21-associated disorders,” “GLP-1-associated disorders,” “Exendin-4-associated disorders,” and terms similarly used herein, includes obesity, type 1 and type 2 diabetes mellitus, pancreatitis, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, acute myocardial infarction, hypertension, cardiovascular disease, atherosclerosis, peripheral arterial disease, stroke, heart failure, coronary heart disease, kidney disease, diabetic complications, neuropathy, gastroparesis, disorders associated with severe inactivating mutations in the insulin receptor, lipodystrophies including HIV-associated lipodystrophy, and other metabolic disorders.

The term “disorders associated with severe inactivating mutations in the insulin receptor,” and terms similarly used herein, describe conditions in subjects afflicted with mutations in the insulin receptor (or possible proteins directly downstream from it) which cause severe insulin resistance but are often (though not always) seen without the obesity common in Type 2 diabetes mellitus. In many ways, subjects afflicted with these conditions manifest hybrid symptoms of Type 1 diabetes mellitus and Type 2 diabetes mellitus. Subjects thereby afflicted fall into several categories of roughly increasing severity, including: Type A Insulin Resistance, Type C Insulin Resistance (AKA HAIR-AN Syndrome), Rabson-Mendenhall Syndrome and finally Donohue's Syndrome or Leprechaunism. These disorders are associated with very high endogenous insulin levels, and very often, hyperglycemia. Subjects thereby afflicted also present with various clinical features associated with “insulin toxicity,” including hyperandrogenism, polycystic ovarian syndrome (PCOS), hirsuitism, and acanthosis nigricans (excessive growth and pigmentation) in the folds of the skin.

“Lipodystrophies, including HIV-associated lipodystrophy” are disorders of adipose tissue characterized by a selective loss of body fat. Patients with lipodystrophy have a tendency to develop insulin resistance, hyperinsulinemia, hyperglycemia, hypertriglyceridemia and fatty liver. There are numerous forms of lipodystrophy that are inherited (genetic) or acquired. The genetic forms of lipodystrophy include congenital generalized lipodystrophy (the Berardinelli-Seip syndrome) and several types of familial partial lipodystrophy (the Dunnigan type, the Kobberling type, the mandibuloacral dysplasis type). The acquired forms of lipodystrophy include acquired generalized lipodystrophy (the Lawrence syndrome), acquired partial lipodystrophy (the Barraquer-Simons syndrome), and lipodystrophy induced by protease inhibitors and nucleoside reverse transcriptase inhibitors used to treat HIV.

“Type 2 diabetes mellitus” is a condition characterized by excess glucose production in spite of the availability of insulin, and circulating glucose levels remain excessively high as a result of inadequate glucose clearance.

“Type 1 diabetes mellitus” is a condition characterized by high blood glucose levels caused by total lack of insulin. This occurs when the body's immune system attacks the insulin-producing beta cells in the pancreas and destroys them. The pancreas then produces little or no insulin.

“Glucose intolerance” or Impaired Glucose Tolerance (IGT) is a pre-diabetic state of dysglycemia that is associated with increased risk of cardiovascular pathology. The pre-diabetic condition prevents a subject from moving glucose into cells efficiently and utilizing it as an efficient fuel source, leading to elevated glucose levels in blood and some degree of insulin resistance.

“Hyperglycemia” is defined as an excess of sugar (glucose) in the blood.

“Hypoglycemia”, also called low blood sugar, occurs when your blood glucose level drops too low to provide enough energy for your body's activities.

“Hyperinsulinemia” is defined as a higher-than-normal level of insulin in the blood.

“Insulin resistance” is defined as a state in which a normal amount of insulin produces a subnormal biologic response.

“Obesity,” in terms of the human subject, can be defined as that body weight over 20 percent above the ideal body weight for a given population (R. H. Williams, Textbook of Endocrinology, 1974, p. 904-916).

“Diabetic complications” are problems, caused by high blood glucose levels, with other body functions such as kidneys, nerves (neuropathies), feet (foot ulcers and poor circulation) and eyes (e.g. retinopathies). Diabetes also increases the risk for heart disease and bone and joint disorders. Other long-term complications of diabetes include skin problems, digestive problems, sexual dysfunction and problems with teeth and gums.

“Metabolic syndrome” can be defined as a cluster of at least three of the following signs: abdominal fat—in most men, a 40-inch waist or greater; high blood sugar—at least 110 milligrams per deciliter (mg/dl) after fasting; high triglycerides—at least 150 mg/dL in the bloodstream; low HDL—less than 40 mg/dl; and, blood pressure of 130/85 mmHg or higher.

“Pancreatitis” is inflammation of the pancreas.

“Dyslipidemia” is a disorder of lipoprotein metabolism, including lipoprotein overproduction or deficiency. Dyslipidemias may be manifested by elevation of the total cholesterol, low-density lipoprotein (LDL) cholesterol and triglyceride concentrations, and a decrease in high-density lipoprotein (HDL) cholesterol concentration in the blood.

“Nonalcoholic fatty liver disease (NAFLD)” is a liver disease, not associated with alcohol consumption, characterized by fatty change of hepatocytes.

“Nonalcoholic steatohepatitis (NASH)” is a liver disease, not associated with alcohol consumption, characterized by fatty change of hepatocytes, accompanied by intralobular inflammation and fibrosis.

“Hypertension” or high blood pressure that is a transitory or sustained elevation of systemic arterial blood pressure to a level likely to induce cardiovascular damage or other adverse consequences. Hypertension has been arbitrarily defined as a systolic blood pressure above 140 mmHg or a diastolic blood pressure above 90 mmHg.

“Cardiovascular diseases” are diseases related to the heart or blood vessels.

“Acute myocardial infarction” occurs when there is interruption of the blood supply to a part of the heart. The resulting ischemia and oxygen shortage, if left untreated for a sufficient period of time, can cause damage or death (infarction) of the heart muscle tissue (myocardium).

“Peripheral arterial disease” occurs when plaque builds up in the arteries that carry blood to the head, organs and limbs. Over time, plaque can harden and narrow the arteries which limits the flow of oxygen-rich blood to organs and other parts of the body.

“Atherosclerosis” is a vascular disease characterized by irregularly distributed lipid deposits in the intima of large and medium-sized arteries, causing narrowing of arterial lumens and proceeding eventually to fibrosis and calcification. Lesions are usually focal and progress slowly and intermittently. Limitation of blood flow accounts for most clinical manifestations, which vary with the distribution and severity of lesions.

“Stroke” is any acute clinical event, related to impairment of cerebral circulation, that lasts longer than 24 hours. A stroke involves irreversible brain damage, the type and severity of symptoms depending on the location and extent of brain tissue whose circulation has been compromised.

“Heart failure”, also called congestive heart failure, is a condition in which the heart can no longer pump enough blood to the rest of the body.

“Coronary heart disease”, also called coronary artery disease, is a narrowing of the small blood vessels that supply blood and oxygen to the heart.

“Kidney disease” or nephropathy is any disease of the kidney. Diabetic nephropathy is a major cause of morbidity and mortality in people with type 1 or type 2 diabetes mellitus.

“Neuropathies” are any diseases involving the cranial nerves or the peripheral or autonomic nervous system.

“Gastroparesis” is weakness of gastric peristalsis, which results in delayed emptying of the bowels.

“Click chemistry” is a term that was introduced by K. B. Sharpless in 2001 to describe reactions that are high yielding, wide in scope, create only byproducts that can be removed without chromatography, are stereospecific, simple to perform, and can be conducted in easily removable or benign solvents.

The critically ill patients encompassed by the present invention generally experience an unstable hypermetabolic state. This unstable metabolic state is due to changes in substrate metabolism, which may lead to relative deficiencies in some nutrients. Generally there is an increased oxidation of both fat and muscle.

Moreover, critically ill patients are preferably patients that experience systemic inflammatory response syndrome or respiratory distress. A reduction in morbidity means reducing the likelihood that a critically ill patient will develop additional illnesses, conditions, or symptoms or reducing the severity of additional illnesses, conditions, or symptoms. For example reducing morbidity may correspond to a decrease in the incidence of bacteremia or sepsis or complications associated with multiple organ failure.

As used herein, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, reference to “an antibody” includes a mixture of two or more such antibodies.

As used herein, the term “about” refers to +1-20%, more preferably, +1-10%, or still more preferably, +1-5% of a value.

The terms “polypeptide” and “protein”, are used interchangeably and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, naturally and non-naturally occurring amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.

The terms “individual”, “subject”, “host” and “patient” are used interchangeably and refer to any subject for whom diagnosis, treatment, or therapy is desired, particularly humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and the like. In some preferred embodiments the subject is a human.

As used herein, the term “sample” refers to biological material from a patient. The sample assayed by the present invention is not limited to any particular type.

Samples include, as non-limiting examples, single cells, multiple cells, tissues, tumors, biological fluids, biological molecules, or supernatants or extracts of any of the foregoing. Examples include tissue removed for biopsy, tissue removed during resection, blood, urine, lymph tissue, lymph fluid, cerebrospinal fluid, mucous, and stool samples. The sample used will vary based on the assay format, the detection method and the nature of the tumors, tissues, cells or extracts to be assayed. Methods for preparing samples are well known in the art and can be readily adapted in order to obtain a sample that is compatible with the method utilized.

As used herein, the term “biological molecule” includes, but is not limited to, polypeptides, nucleic acids, and saccharides.

As used herein, the term “modulating” refers to a change in the quality or quantity of a gene, protein, or any molecule that is inside, outside, or on the surface of a cell. The change can be an increase or decrease in expression or level of the molecule. The term “modulates” also includes changing the quality or quantity of a biological function/activity including, without limitation, the ability to lower blood glucose, insulin, triglyceride, or cholesterol levels; to reduce liver lipid or liver triglyceride levels; to reduce body weight; and to improve glucose tolerance, energy expenditure, or insulin sensitivity.

As used herein, the term “modulator” refers to a composition that modulates one or more physiological or biochemical events associated with an FGF21-associated disorder, such as type 1 or type 2 diabetes mellitus or a metabolic condition like obesity. Said events include but are not limited to the ability to lower blood glucose, insulin, triglyceride, or cholesterol levels; to reduce liver lipid or liver triglyceride levels; to reduce body weight; and to improve glucose tolerance, energy expenditure, or insulin sensitivity.

A “gene product” is a biopolymeric product that is expressed or produced by a gene. A gene product may be, for example, an unspliced RNA, an mRNA, a splice variant mRNA, a polypeptide, a post-translationally modified polypeptide, a splice variant polypeptide etc. Also encompassed by this term are biopolymeric products that are made using an RNA gene product as a template (i.e. cDNA of the RNA). A gene product may be made enzymatically, recombinantly, chemically, or within a cell to which the gene is native. In some embodiments, if the gene product is proteinaceous, it exhibits a biological activity. In some embodiments, if the gene product is a nucleic acid, it can be translated into a proteinaceous gene product that exhibits a biological activity.

“Modulation of FGF21 activity,” “Modulation of GLP-1 activity,” and “Modulation of Exendin-4 activity,” as used herein, refers to an increase or decrease in FGF21, GLP-1, or Exendin-4 activity, respectively, that can be a result of, for example, interaction of an agent with an FGF21, GLP-1, or Exendin-4 polynucleotide or polypeptide, inhibition of FGF21, GLP-1, or Exendin-4 transcription and/or translation (e.g., through antisense or siRNA interaction with the FGF21, GLP-1, or Exendin-4 gene or FGF21, GLP-1, or Exendin-4 transcript, through modulation of transcription factors that facilitate FGF21, GLP-1, or Exendin-4 expression), and the like. For example, modulation of a biological activity refers to an increase or a decrease in a biological activity. FGF21, GLP-1, or Exendin-4 activity can be assessed by means including, without limitation, assaying blood glucose, insulin, triglyceride, or cholesterol levels in a subject, assessing FGF21, GLP-1, or Exendin-4 polypeptide levels, or by assessing FGF21, GLP-1, or Exendin-4 transcription levels.

Comparisons of FGF21, GLP-1, or Exendin-4 activity can also be accomplished by, e.g., measuring levels of an FGF21, GLP-1, or Exendin-4 downstream biomarker, and measuring increases in FGF21, GLP-1, or Exendin-4 signaling. FGF21, GLP-1, or Exendin-4 activity can also be assessed by measuring: cell signaling; kinase activity; glucose uptake into adipocytes; blood insulin, triglyceride, or cholesterol level fluctuations; liver lipid or liver triglyceride level changes; interactions between FGF21, GLP-1, or Exendin-4 and a receptor; or phosphorylation of an FGF21, GLP-1, or Exendin-4 receptor. In some embodiments phosphorylation of an FGF21, GLP-1, or Exendin-4 receptor can be tyrosine phosphorylation. In some embodiments modulation of FGF21, GLP-1, or Exendin-4 activity can cause modulation of an FGF21, GLP-1, or Exendin-4-related phenotype.

A “FGF21, GLP-1, or Exendin-4 downstream biomarker,” as used herein, is a gene or gene product, or measurable indicia of a gene or gene product. In some embodiments, a gene or activity that is a downstream marker of FGF21, GLP-1, or Exendin-4 exhibits an altered level of expression. In some embodiments, an activity of the downstream marker is altered in the presence of an FGF21, GLP-1, or Exendin-4 modulator. In some embodiments, the downstream markers exhibit altered levels of expression when FGF21, GLP-1, or Exendin-4 signaling is perturbed with dual function protein of the present invention. For example, FGF21 downstream markers include, without limitation, glucose or 2-deoxy-glucose uptake, pERK and other phosphorylated or acetylated proteins or NAD levels.

As used herein, the term “up-regulates” refers to an increase, activation or stimulation of an activity or quantity. For example, FGF21, GLP-1, or Exendin-4 modulators, such as the dual function proteins of the invention, may increase or agonize the activity of an FGF21, GLP-1, or Exendin-4 receptor. In one embodiment, one or more of FGFR-1(IIIc), FGFR-2(IIIc), or FGFR-3(IIIc) and/or β-klotho may be up-regulated in response to a dual function protein of the invention. Up-regulation can also refer to an FGF21, GLP-1, or Exendin-4-related activity, such as e.g., the ability to lower blood glucose, insulin, triglyceride, or cholesterol levels; to reduce liver lipid or triglyceride levels; to reduce body weight; to improve glucose tolerance, energy expenditure, or insulin sensitivity; or to cause phosphorylation of an FGF21, GLP-1, or Exendin-4 receptor; or to increase an FGF21, GLP-1, or Exendin-4 downstream marker. For example, the FGFR21 receptor can be one or more of FGFR-1(IIIc), FGFR-2(IIIc), or FGFR-3(IIIc) and/or β-klotho. Up-regulation may range anywhere from 25% to 500% as compared to a control.

As used herein, the term “N-terminus” refers to at least the first 20 amino acids of a protein.

As used herein, the terms “N-terminal domain” and “N-terminal region” are used interchangeably and refer to a fragment of a protein that begins at the first amino acid of the protein and ends at any amino acid in the N-terminal half of the protein. For example, the N-terminal domain of FGF21 is from amino acid 1 of SEQ ID NO:1 to any amino acid between about amino acids 10 and 105 of SEQ ID NO:1.

As used herein, the term “C-terminus” refers to at least the last 20 amino acids of a protein.

As used herein, the terms “C-terminal domain” and “C-terminal region” are used interchangeably and refer to a fragment of a protein that begins at any amino acid in the C-terminal half of the protein and ends at the last amino acid of the protein. For example, the C-terminal domain of FGF21 begins at any amino acid from amino acid 105 to about amino acid 200 of SEQ ID NO:1 and ends at amino acid 209 of SEQ ID NO:1.

The term “domain” as used herein refers to a structural part of a biomolecule that contributes to a known or suspected function of the biomolecule. Domains may be co-extensive with regions or portions thereof and may also incorporate a portion of a biomolecule that is distinct from a particular region, in addition to all or part of that region.

As used herein, the term “signal domain” (also called “signal sequence” or “signal peptide”) refers to a peptide domain that resides in a continuous stretch of amino acid sequence at the N-terminal region of a precursor protein (often a membrane-bound or secreted protein) and is involved in post-translational protein transport. In many cases the signal domain is removed from the full-length protein by specialized signal peptidases after the sorting process has been completed. Each signal domain specifies a particular destination in the cell for the precursor protein. The signal domain of FGF21 is amino acids 1-28 of SEQ ID NO:1.

As used herein, the term “receptor binding domain” refers to any portion or region of a protein that contacts a membrane-bound receptor protein, resulting in a cellular response, such as a signaling event.

As used herein, the term “ligand binding domain” refers to any portion or region of a fusion protein of the invention retaining at least one qualitative binding activity of a corresponding native sequence.

The term “region” refers to a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein. In some embodiments a “region” is associated with a function of the biomolecule.

The term “fragment” as used herein refers to a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a portion is defined by a contiguous portion of the amino acid sequence of that protein and refers to at least 3-5 amino acids, at least 8-10 amino acids, at least 11-15 amino acids, at least 17-24 amino acids, at least 25-30 amino acids, and at least 30-45 amino acids. In the case of oligonucleotides, a portion is defined by a contiguous portion of the nucleic acid sequence of that oligonucleotide and refers to at least 9-15 nucleotides, at least 18-30 nucleotides, at least 33-45 nucleotides, at least 48-72 nucleotides, at least 75-90 nucleotides, and at least 90-130 nucleotides. In some embodiments, portions of biomolecules have a biological activity. In the context of the present invention, FGF21 polypeptide fragments do not comprise the entire FGF21 polypeptide sequence set forth in SEQ ID NO:1.

A “native sequence” polypeptide is one that has the same amino acid sequence as a polypeptide derived from nature. Such native sequence polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. Thus, a native sequence polypeptide can have the amino acid sequence of naturally occurring human polypeptide, murine polypeptide, or polypeptide from any other mammalian species.

As used herein, the phrase “homologous nucleotide sequence,” or “homologous amino acid sequence,” or variations thereof, refers to sequences characterized by a homology, at the nucleotide level or amino acid level, of at least a specified percentage and is used interchangeably with “sequence identity.” Homologous nucleotide sequences include those sequences coding for isoforms of proteins. Such isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. Homologous nucleotide sequences include nucleotide sequences encoding for a protein of a species other than humans, including, but not limited to, mammals. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. Homologous amino acid sequences include those amino acid sequences which contain conservative amino acid substitutions and which polypeptides have the same binding and/or activity. In some embodiments, a nucleotide or amino acid sequence is homologous if it has at least 60% or greater, up to 99%, identity with a comparator sequence. In some embodiments, a nucleotide or amino acid sequence is homologous if it shares one or more, up to 60, nucleotide/amino acid substitutions, additions, or deletions with a comparator sequence. In some embodiments, the homologous amino acid sequences have no more than 5 or no more than 3 conservative amino acid substitutions.

Percent homology or identity can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). In some embodiments, homology between the probe and target is between about 75% to about 85%. In some embodiments, nucleic acids have nucleotides that are at least about 95%, about 97%, about 98%, about 99% and about 100% homologous to SEQ ID NO:2, or a portion thereof.

Homology may also be at the polypeptide level. In some embodiments, constituent polypeptides of the dual function proteins of the invention may be at least 95% homologous to their full-length wild-type counterparts or corresponding native sequences, or to portions thereof. The degree or percentage identity of dual function proteins of the invention, or portions thereof, and different amino acid sequences is calculated as the number of exact matches in an alignment of the two sequences divided by the length of the “invention sequence” or the “foreign sequence”, whichever is shortest. The result is expressed as percent identity.

As used herein, the term “mixing” refers to the process of combining one or more compounds, cells, molecules, and the like together in the same area. This may be performed, for example, in a test tube, petri dish, or any container that allows the one or more compounds, cells, or molecules, to be mixed.

As used herein, the term “substantially purified” refers to a compound (e.g., either a polynucleotide or a polypeptide or an antibody) that is removed from its natural environment and is at least 60% free, at least 75% free, and at least 90% free from other components with which it is naturally associated.

The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which can be administered without undue toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in therapeutic compositions can include liquids such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, can also be present in such vehicles.

Naturally occurring disulfide bonds, as provided by cysteine residues, generally increase thermodynamic stability of proteins. Successful examples of increased thermodynamic stability, as measured in increase of the melting temperature, are multiple disulfide-bonded mutants of the enzymes T4 lysozyme (Matsumura et al., PNAS 86:6562-6566 (1989)) and barnase (Johnson et al., J. Mol. Biol. 268:198-208 (1997)). An aspect of the present invention is an enhancement of the physical stability of FGF21 in the presence of a preservative, achieved by the presence of disulfide bonds within the variants, which constrain the flexibility of wild-type FGF21 and thereby limit access of the preservative to the hydrophobic core of the protein. Enhancement of the physical stability of FGF21 within the proteins of the present invention, due to the presence of disulfide bonds within the variants, can confer additional protection to said proteins, whether or not in the presence of a preservative, including but not limited to protection against fluctuations in environmental conditions such as pH and temperature.

Improvements of the Dual Function Proteins of the Invention Over Wild Type Protein Comparators and Combinations Thereof

It is well known in the art that a significant challenge in the development of protein pharmaceuticals is to deal with the physical and chemical instabilities of proteins.

This is even more apparent when a protein pharmaceutical formulation is intended to be a multiple use, injectable formulation requiring a stable, concentrated and preserved solution, while maintaining a favorable bioactivity profile. Biophysical characterization of wild-type FGF21 in the literature established that a concentrated protein solution (>5 mg/ml), when exposed to stress conditions, such as high temperature or low pH, lead to accelerated association and aggregation (i.e., poor physical stability and biopharmaceutical properties). Exposure of a concentrated protein solution of FGF21 to pharmaceutical preservatives (e.g., m-cresol) also had a negative impact on physical stability.

Therefore, an embodiment of the present invention is to enhance physical stability of concentrated solutions, while maintaining chemical stability and biological potency, under both physiological and preserved formulation conditions. It is thought that association and aggregation may result from hydrophobic interactions, since, at a given protein concentration, temperature, and ionic strength have considerable impact on physical stability.

For the most part, non-conserved, presumed surface exposed amino acid residues are targeted. The local environment of these residues is analyzed and, those not deemed structurally important are selected for mutagenesis. One method to initiate specific changes is to further decrease the pl of the protein by introducing glutamic acid residues (“glutamic acid scan”). The introduction of charged substitutes can inhibit hydrophobic-mediated aggregation via charge-charge repulsion and potentially improve preservative compatibility. In addition, one skilled in the art would also recognize that with sufficient mutagenesis, the pl could be shifted into a basic pH range by the introduction of positive charge, with or without concomitant decrease in negative charge, thus allowing for charge-charge repulsion.

An additional difficulty associated with therapeutic applications of wild-type FGF21 as a biotherapeutic, for instance, is that its half-life is very short in vivo (on the order of 0.5 and 2 h, respectively, in mouse and primate). There is hence a need to develop follow-up compounds that are more efficacious either through higher potency or longer half-life. Similarly, various mechanisms have been employed to enhance serum half-life for GLP-1 (1-2 minutes due to rapid cleavage by endogenous proteases, particularly dipeptidyl peptidase-4 (DPP4)) through use of Exendin-4 or other analogues that are resistant to cleavage by DPP4 as well as further modifications and formulations for extended half-life or slow release of compound.

The proteins of the invention, e.g., dual FGF21 receptor agonist and GLP-1 receptor agonist proteins of the invention, were developed as a way to achieve the desirable effects of FGF21 and GLP-1 treatment at a higher potency and in a half-life-extended formulation. The co-administration of GLP-1 and FGF21 has been described in the literature, e.g., in patent publications WO2010/068735 and WO2009/020802, with data suggesting additive or synergistic effects of co-administration of GLP-1 and FGF21 for treatment of obesity and type 2 diabetes. However, co-localization of the two receptor agonists in a single molecule, e.g., in the form of the proteins of the invention, provides better access of both GLP-1 and FGF21 to tissues or cells and provides increased benefit over simple co-administration as separate entities. This advantage of combining two agonists into a single molecule compared to simple co-administration of each is especially advantageous for tissues in which both the GLP-1 and the FGF21 receptors are expressed in the same tissues, such as adipose, pancreatic β-cells, hepatic and hypothalamic cells. Dual FGF21 receptor agonist and GLP-1 receptor agonist proteins of the invention and other single dual activity entities have improved biological properties due to altered receptor trafficking, altered signal transduction effects and/or entropic (avidity) effects.

Because FGF21 and GLP-1 (or Exendin-4) act through different mechanisms, it is expected that they will have additive or synergistic effects when, for example, administered at the same time in the form of the dual function proteins of the invention. GLP-1 and Exendin-4 act primarily to increase insulin secretion in response to food intake while FGF21 sensitizes the body to respond better to insulin, which may provide benefit for both type 1 and type 2 diabetes in managing blood glucose levels. Beta-cell protective effects combined with the improved beta-cell function and insulin sensitivity has the potential to provide benefit even in type 1 diabetics.

Another example is weight loss: The satiety signal and slowed gastric emptying by GLP-1 or Exendin-4 are expected to lower appetite while FGF21 increases metabolism in adipose and other tissues which could increase loss of fat. These two effects could result in an additive or even synergistic weight loss with combined dosing. Expression of receptors for both FGF21 and GLP-1 in metabolically active tissues suggests that simultaneous delivery of a FGF21 receptor agonist and a GLP-1 receptor agonist to such tissues may be beneficial for treating metabolic diseases. A combination of GLP-1 and Exendin-4's cardioprotective effects and improved lipid profiles seen with FGF21 could also result in an additive benefit for cardiovascular disease associated with obesity, type 2 and type 1 diabetes.

Another likely benefit of GLP-1 and FGF21 dual agonism is an improved, i.e., reduced, side effect profile. Recent studies (Wei et al. (2012) PNAS 109, 3143-48) indicate that treatment of diet-induced obese mice with FGF21 induces bone loss, due to a diminished inactivation of PPARγ (via reduced sumoylation); a shift of mesenchymal stem cell differentiation from osteoblasts to adipocytes is seen in the presence of increased PPARγ activity in the bone following FGF21 treatment. However, it has also been reported (Sanz et al. (2010) Am J Physiol Endocrinol Metab 298, E634-E643) that GLP-1 can reduce the differentiation of human mesenchymal stem cells to adipocytes by reducing the expression of PPARγ. Therefore, as GLP-1 has the potential to be bone protective, GLP-1-FGF21 fusion proteins are likely to reduce the risk of bone loss compared to FGF21-only treatments.

Although the embodiments of the present invention concern the physical and chemical stability under both physiological and preserved pharmaceutical formulation conditions, maintaining the biological potency of the proteins of the invention as compared to, e.g., wild-type FGF21 is an important factor of consideration as well. Therefore, the biological potency of the proteins of the present invention is defined by the ability of the proteins to affect glucose uptake and/or the lowering of plasma glucose levels, as shown herein in the examples.

The proteins, polypeptides, and/or peptides of the invention administered according to this invention may be generated and/or isolated by any means known in the art. The most preferred method for producing the variant is through recombinant DNA methodologies and is well known to those skilled in the art. Such methods are described in Current Protocols in Molecular Biology (John Wiley & Sons, Inc.), which is incorporated herein by reference.

Additionally, the preferred embodiments include a biologically active peptide derived from the variant described herein. Such a peptide will contain at least one of the substitutions described and the variant will possess biological activity. The peptide may be produced by any and all means known to those skilled in the art, examples of which included but are not limited to enzymatic digestion, chemical synthesis or recombinant DNA methodologies.

It is established in the art that fragments of peptides of certain fibroblast growth factors are biologically active. See for example, Baird et al., Proc. Natl. Acad. Sci (USA) 85:2324-2328 (1988), and J. Cell. Phys. Suppl. 5:101-106 (1987). Therefore, the selection of fragments or peptides of the variant is based on criteria known in the art. For example, it is known that dipeptidyl peptidase IV (DPP-IV, or DPP-4) is a serine type protease involved in inactivation of neuropeptides, endocrine peptides, and cytokines (Damme et al. Chem. Immunol. 72: 42-56, (1999)). The N-terminus of FGF21 (HisProllePro) contains two dipeptides that could potentially be substrates to DPP-IV, resulting in a fragment of FGF21 truncated at the N-terminus by 4 amino acids. Unexpectedly, this fragment of wild-type FGF21 has been demonstrated to retain biological activity, thus, proteins of the present invention truncated at the N-terminus by up to 4 amino acids, is an embodiment of the present invention.

The invention also encompasses polynucleotides encoding the above-described variants that may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded. The coding sequences that encode the proteins of the present invention may vary as a result of the redundancy or degeneracy of the genetic code.

The polynucleotides that encode for the proteins of the invention may include the following: only the coding sequence for the variant, the coding sequence for the variant and additional coding sequence such as a functional polypeptide, or a leader or secretory sequence or a pro-protein sequence; the coding sequence for the variant and non-coding sequence, such as introns or non-coding sequence 5′ and/or 3′ of the coding sequence for the variant. Thus the term “polynucleotide encoding a variant” encompasses a polynucleotide that may include not only coding sequence for the variant but also a polynucleotide, which includes additional coding and/or non-coding sequence.

The invention further relates to variants of the described polynucleotides that encode for fragments, analogs and derivatives of the polypeptide that contain the indicated substitutions. The variant of the polynucleotide may be a naturally occurring allelic variant of the human FGF21 sequence, a non-naturally occurring variant, or a truncated variant as described above. Thus, the present invention also includes polynucleotides encoding the variants described above, as well as variants of such polynucleotides, which variants encode for a fragment, derivative or analog of the disclosed variant. Such nucleotide variants include deletion variants, substitution variants, truncated variants, and addition or insertion variants as long as at least one of the indicated amino acid substitutions of the first or second embodiments is present.

The polynucleotides of the invention will be expressed in hosts after the sequences have been operably linked to (i.e., positioned to ensure the functioning of) an expression control sequence. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors will contain selection markers, e.g., tetracycline, neomycin, and dihydrofolate reductase, to permit detection of those cells transformed with the desired DNA sequences. The dual function proteins or fragments thereof can be expressed in mammalian cells, insect, yeast, bacterial or other cells under the control of appropriate promoters. Cell free translation systems can also be employed to produce such proteins using RNAs derived from DNA constructs of the present invention.

E. coli is a prokaryotic host useful particularly for cloning the polynucleotides of the present invention. Other microbial hosts suitable for use include Bacillus subtilus, Salmonella typhimurium, and various species of Serratia, Pseudomonas, Streptococcus, and Staphylococcus, although others may also be employed as a matter of choice. In these prokaryotic hosts, one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any of a number of well-known promoters may be present, such as the lactose promoter system, a tryptophan (Trp) promoter system, a beta-lactamase promoter system, or a promoter system from phages lambda or T7. The promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation.

One skilled in the art of expression of proteins will recognize that methionine or methionine-arginine sequence can be introduced at the N-terminus of the mature sequence (SEQ ID NO: 3) for expression in E. coli and are contemplated within the context of this invention. Thus, unless otherwise noted, proteins of the present invention expressed in E. coli have a methionine sequence introduced at the N-terminus.

Other microbes, such as yeast or fungi, may also be used for expression. Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia angusta are examples of preferred yeast hosts, with suitable vectors having expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired. Aspergillus niger, Trichoderma reesei; and Schizophyllum commune, are examples of fungi hosts, although others may also be employed as a matter of choice.

Mammalian tissue cell culture may also be used to express and produce the polypeptides of the present invention. Eukaryotic cells are actually preferred, because a number of suitable host cell lines capable of secreting intact variants have been developed in the art, and include the CHO cell lines, various COS cell lines, NSO cells, Syrian Hamster Ovary cell lines, HeLa cells, or human embryonic kidney cell lines (i.e. HEK293, HEK293EBNA).

Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from SV40, adenovirus, bovine papilloma virus, cytomegalovirus, Raus sarcoma virus, and the like. Preferred polyadenylation sites include sequences derived from SV40 and bovine growth hormone.

The vectors containing the polynucleotide sequences of interest (e.g., the Proteins of the invention and expression control sequences) can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts.

Various methods of protein purification may be employed and such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology 182: 83-9 (1990) and Scopes, Protein Purification: Principles and Practice, Springer-Verlag, NY (1982). The purification step(s) selected will depend, for example, on the nature of the production process used for the Proteins of the invention.

The proteins, polypeptides, and/or peptides of the invention, e.g., the dual activity fusion proteins of the invention, should be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the patient, the site of delivery of the protein compositions, the method of administration, the scheduling of administration, and other factors known to practitioners. The “therapeutically effective amount” of the proteins of the invention for purposes herein is thus determined by such considerations.

The pharmaceutical compositions of the proteins of the present invention may be administered by any means that achieve the generally intended purpose: to treat type 1 and type 2 diabetes mellitus, obesity, metabolic syndrome, or critically ill patients. Non-limiting permissible means of administration include, for example, by inhalation or suppository or to mucosal tissue such as by lavage to vaginal, rectal, urethral, buccal and sublingual tissue, orally, nasally, topically, intranasally, intraperitoneally, parenterally, intravenously, intramuscularly, intrasternally, by intraarticular injection, intralymphatically, interstitially, intra-arterially, subcutaneously, intrasynovial, transepithelial, and transdermally. In some embodiments, the pharmaceutical compositions are administered by lavage, orally or inter-arterially. Other suitable methods of introduction can also include rechargeable or biodegradable devices and slow or sustained release polymeric devices. The pharmaceutical compositions of this invention can also be administered as part of a combinatorial therapy with other known metabolic agents.

The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. Compositions within the scope of the invention include all compositions wherein an FGF21 variant is present in an amount that is effective to achieve the desired medical effect for treatment type 1 or type 2 diabetes mellitus, obesity, or metabolic syndrome. While individual needs may vary from one patient to another, the determination of the optimal ranges of effective amounts of all of the components is within the ability of the clinician of ordinary skill.

The proteins of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions. A desired formulation would be one that is a stable lyophilized product that is reconstituted with an appropriate diluent or an aqueous solution of high purity with optional pharmaceutically acceptable carriers, preservatives, excipients or stabilizers [Remington's Pharmaceutical Sciences 16th edition (1980)]. The proteins of the present invention may be combined with a pharmaceutically acceptable buffer, and the pH adjusted to provide acceptable stability, and a pH acceptable for administration.

For parenteral administration, in one embodiment, the proteins of the invention are formulated generally by mixing one or more of them at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. Preferably, one or more pharmaceutically acceptable anti-microbial agents may be added. Phenol, m-cresol, and benzyl alcohol are preferred pharmaceutically acceptable anti-microbial agents.

Optionally, one or more pharmaceutically acceptable salts may be added to adjust the ionic strength or tonicity. One or more excipients may be added to further adjust the isotonicity of the formulation. Glycerin, sodium chloride, and mannitol are examples of an isotonicity adjusting excipient.

Those skilled in the art can readily optimize pharmaceutically effective dosages and administration regimens for therapeutic compositions comprising Proteins of the invention, as determined by good medical practice and the clinical condition of the individual patient. A typical dose range for the proteins of the present invention will range from about 0.01 mg per day to about 1000 mg per day (or about 0.05 mg per week to about 5000 mg per week administered once per week) for an adult. Preferably, the dosage ranges from about 0.1 mg per day to about 100 mg per day (or about 0.5 mg per week to about 500 mg per week administered once per week), more preferably from about 1.0 mg/day to about 10 mg/day (or about 5 mg per week to about 50 mg per week administered once per week). Most preferably, the dosage is about 1-5 mg/day (or about 5 mg per week to about 25 mg per week administered once per week). The appropriate dose of an FGF21 variant administered will result in lowering blood glucose levels and increasing energy expenditure by faster and more efficient glucose utilization, and thus is useful for treating type 1 and type 2 diabetes mellitus, obesity and metabolic syndrome.

In addition, because hyperglycemia and insulin resistance are common in critically ill patients given nutritional support, some intensive care units (ICUs) administer insulin to treat excessive hyperglycemia in fed critically ill patients. In fact, recent studies document the use of exogenous insulin to maintain blood glucose at a level no higher than 110 mg per deciliter reduced morbidity and mortality among critically ill patients in the surgical intensive care unit, regardless of whether they had a history of diabetes (Van den Berghe et al. N Engl J Med., 345(19):1359, (2001)). Thus, proteins of the present invention are uniquely suited to help restore metabolic stability in metabolically unstable critically ill patients. Proteins of the invention such as those containing variants of FGF21 are unique in that they stimulate glucose uptake and enhances insulin sensitivity but do not induce hypoglycemia.

In another aspect of the present invention, proteins of the invention for use as a medicament for the treatment of obesity, type 1 and type 2 diabetes mellitus, pancreatitis, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, acute myocardial infarction, conditions associated with severe inactivating mutations in the insulin receptor, lipodystrophies including HIV-associated lipodystrophy, and other metabolic disorders is contemplated.

Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications); and Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989).

Site-Specific FGF21 Mutants

In some embodiments, the fusion proteins of the invention include additional mutants of GLP-1 or Exendin-4, GLP-1/Glucagon hybrid peptides, GLP-1 analogues with unnatural amino acids (to convey DPP-4 resistance, for PEGylation, or for other purposes).

In some embodiments, the proteins of the invention comprise FGF21 agonists with one or more of the following additional modifications of wild-type FGF21:

(i) additional disulfides, unnatural amino acids, or modifications to promote dimerization such as formation of a disulfide at R154C or introduction of a cysteine at another site, or dimerization through a fused Fc domain, or dimer formation through a cross-linker such as a bifunctional PEG;

(ii) fragments of FGF21;

(iii) proteins selected to have FGF21 activity (binding to beta-klotho and binding and activation of the FGFR's); and

(iv) an FGF21 mimetic antibody (of various formats such as Fab, unibody, svFc etc.).

In some embodiments, the dual activity proteins of the invention comprise one or more of the following linkers: a simple amide bond, short peptides (particularly Ser/Gly repeats), additional residues from the FGF21 translated sequence, or a larger linker up to an entire protein (such as an Fc domain, an HSA-binding helix bundle, HSA, etc.). The two moieties can also be linked by other chemical means, such as through unnatural amino acids or standard chemical linkers (maleimide-Cys, NHS-Lys, “click chemistry”, etc.) “Linker” for the FGF21 mimetic antibody approach could include those already listed and also an insertion into a loop with subsequent cleavage to release the GLP-1 N-terminus.

In some embodiments, the fusion protein of the invention comprises PEGylation occurring at one, two, or more specific sites. In preferred embodiments, the PEGylation occurs within the FGF21 molecule or the linker. In some embodiments, the PEGylation is not within ˜10 amino acids of the N-terminus of the dual function protein, preferably not within ˜10 amino acids of the start of FGF21. PEGylation attachment chemistries can include NHS-Lys, maleimide-Cys, unnatural amino acids (aldehyde, “click chemistry”, Pcl, etc.) and can be combined with suitable protein variants to control the stoichiometry of the reaction.

The PEG group of the fusion proteins of the invention can be of any size (e.g., 1, 2, 3.4, 5, 10, 20, 24, 29, 30, 40 kDa), and can be linear, branched, or comb structured, with a preference for a total PEGylation of greater than or equal to 40 kDa. Optimal PEGylation achieves half-life extension sufficient for once weekly dosing. PEGylation of protein dimers, trimers, tetramers etc. may result in adequate serum half-life extension using shorter PEG polymers. Branched and comb PEG structures may be beneficial in terms of lower viscosity.

Preferred half-life extension methodologies for the fusion proteins of the invention include integrating an IgG Fc domain or HSA into the linker and may not require PEGylation. Additionally, using Fc domain fusions will result in dimerization and may result in enhanced potency in addition to half-life extension.

Other embodiments of the invention include but are not limited to the following attachments, for half-life extension: HSA-binding lipid or small molecule or micelle to either the monomeric or a dimeric version of the fusion.

In certain embodiments of the invention, other attachments may be made to proteins, polypeptides, and/or peptides of the invention, to achieve half-life extension and other improved biological properties. They can include attaching PEG-cholesterol conjugates (including micelles and liposomes) to the proteins, polypeptides, and/or peptides of the invention, and/or attaching sugars (glycosylate) to the proteins, polypeptides, and/or peptides of the invention. In still other embodiments, similar techniques are employed to add conjugates of, e.g., polysialic acid (PSA), hydroxyethyl starch (HES), albumin-binding ligands, or carbohydrate shields to proteins, polypeptides, and/or peptides.

The HESylation technique, for example, couples branched hydroxyethylstarch (HES) chains (60 kDa or 100 kDa, highly branched amylopectin fragments from corn starch) to a protein, polypeptides, and/or peptides via reductive alkylation. Polysialation conjugates proteins, polypeptides, and/or peptides of interest with polysialic acid (PSA) polymers in a manner similar to PEGylation. PSA polymers are negatively charged, non-immunogenic polymers that occur naturally in the body and are available in molecular weights of 10-50 kD.

In still other embodiments of the invention, other attachments or modifications may be made to proteins, polypeptides, and/or peptides of the invention, to achieve half-life extension and other improved biological properties. These include the creation of recombinant PEG (rPEG) groups, and their attachment to the proteins, polypeptides, and/or peptides of the invention. As developed by the company Amunix, Inc. The rPEG technology is based on protein sequences with PEG-like properties that are genetically fused to biopharmaceuticals, avoiding the extra chemical conjugation step. rPEGs are extended half-life Exenatide constructs that contain a long unstructured tail of hydrophilic amino acids, and which are capable of both increasing a protein or peptide's serum half-life and slowing its rate of absorption, thus reducing the peak-trough ratio significantly. rPEGs have an increased hydrodynamic radius and show an apparent molecular weight that is about 15-fold their actual molecular weight, mimicking the way PEGylation achieves a long serum half-life. Similar recombinant polypeptide sequences are also developed by XL-protein GmbH for the “PASylation” of proteins.

Chemically-Modified Dual Function Protein Mutants

Chemically modified forms of the fusion proteins described herein, including, e.g., truncated and variant forms of the dual function fusion proteins described herein, can be prepared by one skilled in the art, given the disclosures described herein. Such chemically modified dual function proteins are altered such that the chemically modified mutant is different from the unmodified mutant, either in the type or location of the molecules naturally attached to the mutant. Chemically modified mutants can include molecules formed by the deletion of one or more naturally-attached chemical groups.

In one embodiment, proteins of the present invention can be modified by the covalent attachment of one or more polymers. For example, the polymer selected is typically water-soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. Included within the scope of suitable polymers is a mixture of polymers. Preferably, for therapeutic use of the end-product preparation, the polymer will be pharmaceutically acceptable. Non-water soluble polymers conjugated to proteins of the present invention also form an aspect of the invention.

Exemplary polymers each can be of any molecular weight and can be branched or unbranched. The polymers each typically have an average molecular weight of between about 2 kDa to about 100 kDa (the term “about” indicating that in preparations of a water-soluble polymer, some molecules will weigh more and some less than the stated molecular weight). The average molecular weight of each polymer is preferably between about 5 kDa and about 50 kDa, more preferably between about 12 kDa and about 40 kDa, and most preferably between about 20 kDa and about 35 kDa.

Suitable water-soluble polymers or mixtures thereof include, but are not limited to, N-linked or O-linked carbohydrates, sugars, phosphates, polyethylene glycol (PEG) (including the forms of PEG that have been used to derivatize proteins, including mono-(C1-C10), alkoxy-, or aryloxy-polyethylene glycol), monomethoxy-polyethylene glycol, dextran (such as low molecular weight dextran of, for example, about 6 kD), cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), and polyvinyl alcohol. Also encompassed by the present invention are bifunctional crosslinking molecules that can be used to prepare covalently attached dual function protein variant multimers. Also encompassed by the present invention are dual function protein variants covalently attached to polysialic acid.

In some embodiments of the present invention, a dual function protein variant is covalently, or chemically, modified to include one or more water-soluble polymers, including, but not limited to, polyethylene glycol (PEG), polyoxyethylene glycol, or polypropylene glycol. See, e.g., U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; and 4,179,337. In some embodiments of the present invention, adual function protein comprises one or more polymers, including, but not limited to, monomethoxy-polyethylene glycol, dextran, cellulose, another carbohydrate-based polymer, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, or mixtures of such polymers.

In some embodiments of the present invention, a dual function protein is covalently-modified with PEG subunits. In some embodiments, one or more water-soluble polymers are bonded at one or more specific positions (for example, at the N-terminus) of the dual function protein mutant. In some embodiments, one or more water-soluble polymers are randomly attached to one or more side chains of a dual function protein mutant. In some embodiments, PEG is used to improve the therapeutic capacity of a dual function protein mutant. Certain such methods are discussed, for example, in U.S. Pat. No. 6,133,426, which is hereby incorporated by reference for any purpose.

In embodiments of the present invention wherein the polymer is PEG, the PEG group can be of any convenient molecular weight, and can be linear or branched.

The average molecular weight of the PEG group will preferably range from about 2 kD to about 100 kDa, and more preferably from about 5 kDa to about 50 kDa, e.g., 10, 20, 30, 40, or 50 kDa. The PEG groups will generally be attached to the dual function protein mutant via acylation or reductive alkylation through a reactive group on the PEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to a reactive group on the dual function protein mutant (e.g., an aldehyde, amino, or ester group).

Branched PEG derivatives, also known as “Y-shaped” PEG derivatives, contain two linear methoxy PEG chain attached to a central core. The sterically bulky structure of these “Y-shaped” PEG derivatives will facilitate the single point attachment of the modified molecules. By way of example, three kinds of “Y-shaped” PEG derivatives are Y-NHS-40K (useful for amine PEGylation); Y-MAL-40K (useful for thiol PEGylation); and Y-ALD-40K (e.g., Y-AALD-40K and Y-PALD-40K)(useful for N-terminal PEGylation). For amine PEGylation, the “Y-shape” NHS ester will react with the amino group of lysine(s) or the N-terminal amine in biological active molecules to produce a stable amide linkage(s). This NHS ester will couple with the targeted molecules at pH 7-8.5. For thiol PEGylation, the “Y-shape” maleimide will react with the thiol groups in biological active molecules to generate a stable 3-thiosuccinimidyl ether linkage. This maleimide will couple with the targeted molecules at an approximate pH of 7.4 in the presence of other functional groups. For N-terminal PEGylation, the “Y-shape” aldehyde will preferably react with the N-terminal amine in biological active molecules to produce a stable amine linkage in the presence of a reducing reagent such as sodium cyanoborohydride. This aldehyde will couple with the N-terminal amine of the targeted molecules at pH 5-8. Reagents for performing branched PEGylation are available through, e.g., JenKem Technology.

The PEGylation of a polypeptide, including the proteins of the invention, can be specifically carried out using any of the PEGylation reactions known in the art. Such reactions are described, for example, in the following references: Francis et al., 1992, Focus on Growth Factors 3: 4-10; European Patent Nos. 0 154 316 and 0 401 384; and U.S. Pat. No. 4,179,337. For example, PEGylation can be carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer) as described herein. For the acylation reactions, a selected polymer should have a single reactive ester group. For reductive alkylation, a selected polymer should have a single reactive aldehyde group. A reactive aldehyde is, for example, polyethylene glycol propionaldehyde, which is water stable, or mono C1-C10 alkoxy or aryloxy derivatives thereof (see, e.g., U.S. Pat. No. 5,252,714).

In some embodiments of the present invention, a useful strategy for the attachment of the PEG group to a polypeptide involves combining, through the formation of a conjugate linkage in solution, a peptide and a PEG moiety, each bearing a special functionality that is mutually reactive toward the other. The peptides can be easily prepared with conventional solid phase synthesis. The peptides are “preactivated” with an appropriate functional group at a specific site. The precursors are purified and fully characterized prior to reacting with the PEG moiety. Ligation of the peptide with PEG usually takes place in aqueous phase and can be easily monitored by reverse phase analytical HPLC. The PEGylated peptides can be easily purified by preparative HPLC and characterized by analytical HPLC, amino acid analysis and laser desorption mass spectrometry.

Polysaccharide polymers are another type of water-soluble polymer that can be used for protein modification. Therefore, the Proteins of the invention fused to a polysaccharide polymer form embodiments of the present invention. Dextrans are polysaccharide polymers comprised of individual subunits of glucose predominantly linked by alpha 1-6 linkages. The dextran itself is available in many molecular weight ranges, and is readily available in molecular weights from about 1 kD to about 70 kD. Dextran is a suitable water-soluble polymer for use as a vehicle by itself or in combination with another vehicle (e.g., Fc). See, e.g., International Publication No. WO 96/11953. The use of dextran conjugated to therapeutic or diagnostic immunoglobulins has been reported. See, e.g., European Patent Publication No. 0 315 456, which is hereby incorporated by reference. The present invention also encompasses the use of dextran of about 1 kD to about 20 kD.

In general, chemical modification can be performed under any suitable condition used to react a protein with an activated polymer molecule. Methods for preparing chemically modified polypeptides will generally comprise the steps of: (a) reacting the polypeptide with the activated polymer molecule (such as a reactive ester or aldehyde derivative of the polymer molecule) under conditions whereby a FGF21 protein variant becomes attached to one or more polymer molecules, and (b) obtaining the reaction products. The optimal reaction conditions will be determined based on known parameters and the desired result. For example, the larger the ratio of polymer molecules to protein, the greater the percentage of attached polymer molecule. In one embodiment of the present invention, chemically modified FGF21 mutants can have a single polymer molecule moiety at the amino-terminus (see, e.g., U.S. Pat. No. 5,234,784)

In another embodiment of the present invention, proteins of the invention can be chemically coupled to biotin. The biotinylated proteins of the invention are then allowed to bind to avidin, resulting in tetravalent avidin/biotin/proteins of the invention. Proteins of the invention can also be covalently coupled to dinitrophenol (DNP) or trinitrophenol (TNP) and the resulting conjugates precipitated with anti-DNP or anti-TNP-IgM to form decameric conjugates with a valency of 10.

Generally, conditions that can be alleviated or modulated by the administration of the present chemically modified dual function proteins mutants include those described herein for proteins of the invention. However, the chemically modified dual function proteins mutants disclosed herein can have additional activities, enhanced or reduced biological activity, or other characteristics, such as increased or decreased half-life, as compared to unmodified dual function proteins mutants.

Therapeutic Compositions of Dual Function Proteins and Administration Thereof

Therapeutic compositions comprising the dual function proteins of the invention are within the scope of the present invention, and are specifically contemplated in light of, e.g., the identification of several mutant dual function proteins sequences exhibiting enhanced properties. Such dual function proteins mutant pharmaceutical compositions can comprise a therapeutically effective amount of a dual function proteins protein variant in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration.

Acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed.

The pharmaceutical composition can contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides; preferably sodium or potassium chloride; or mannitol sorbitol), delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants (see, e.g., Remington's Pharmaceutical Sciences (18th Ed., A. R. Gennaro, ed., Mack Publishing Company 1990), and subsequent editions of the same, incorporated herein by reference for any purpose).

The optimal pharmaceutical composition will be determined by a skilled artisan depending upon, for example, the intended route of administration, delivery format, and desired dosage (see, e.g., Remington's Pharmaceutical Sciences, supra). Such compositions can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the fusion protein of the invention.

The primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier for injection can be water, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute. In one embodiment of the present invention, dual function pharmaceutical compositions can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. Further, the dual function protein product can be formulated as a lyophilizate using appropriate excipients such as sucrose.

The pharmaceutical compositions containing the fusion proteins of the invention can be selected for parenteral delivery. Alternatively, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art.

The formulation components are present in concentrations that are acceptable to the site of administration. For example, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.

When parenteral administration is contemplated, the therapeutic compositions for use in this invention can be in the form of a pyrogen-free, parenterally acceptable, aqueous solution comprising the desired dual function protein in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which a dual function protein is formulated as a sterile, isotonic solution, properly preserved. Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, that provides for the controlled or sustained release of the product which can then be delivered via a depot injection. Hyaluronic acid can also be used, and this can have the effect of promoting sustained duration in the circulation. Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.

In one embodiment, a pharmaceutical composition can be formulated for inhalation. For example, a dual function protein of the invention can be formulated as a dry powder for inhalation. Dual function protein inhalation solutions can also be formulated with a propellant for aerosol delivery. In yet another embodiment, solutions can be nebulized. Pulmonary administration is further described in International Publication No. WO 94/20069, which describes the pulmonary delivery of chemically modified proteins.

It is also contemplated that certain formulations can be administered orally. In one embodiment of the present invention, dual function proteins of the invention that are administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. For example, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to facilitate absorption of the dual function proteins of the invention. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.

Another pharmaceutical composition can involve an effective quantity of the proteins of the invention in a mixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions can be prepared in unit-dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.

Additional pharmaceutical compositions comprising dual function proteins of the invention will be evident to those skilled in the art, including formulations involving dual function proteins of the invention in sustained- or controlled-delivery formulations. Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art (see, e.g., International Publication No. WO 93/15722, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions, and Wischke & Schwendeman, 2008, Int. J Pharm. 364: 298-327, and Freiberg & Zhu, 2004, Int. J Pharm. 282: 1-18, which discuss microsphere/microparticle preparation and use).

Additional examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and European Patent No. 0 058 481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 22: 547-56), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15: 167-277 and Langer, 1982, Chem. Tech. 12: 98-105), ethylene vinyl acetate (Langer et al., supra) or poly-D-3-hydroxybutyric acid (European Patent No. 0 133 988). Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art. See, e.g., Epstein et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82: 3688-92; and European Patent Nos. 0 036 676, 0 088 046, and 0 143 949.

The pharmaceutical compositions of the invention to be used for in vivo administration typically must be sterile. This can be accomplished by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using this method can be conducted either prior to, or following, lyophilization and reconstitution. The composition for parenteral administration can be stored in lyophilized form or in a solution. In addition, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

Once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.

In a specific embodiment, the present invention is directed to kits for producing a single-dose administration unit. The kits can each contain both a first container having a dried protein and a second container having an aqueous formulation. Also included within the scope of this invention are kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes).

Dosages of Dual Function Proteins and Administration Thereof

The effective amount of a pharmaceutical composition of the invention to be employed therapeutically will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the fusion protein variant is being used, the route of administration, and the size (body weight, body surface, or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect. A typical dosage can range from about 0.1 μg/kg to up to about 100 mg/kg or more, depending on the factors mentioned above. In other embodiments, the dosage can range from 0.1 μg/kg up to about 100 mg/kg; or 1 μg/kg up to about 100 mg/kg.

The frequency of dosing will depend upon the pharmacokinetic parameters of the dual function protein in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition can therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages can be ascertained through use of appropriate dose-response data.

The route of administration of the pharmaceutical composition is in accord with known methods, e.g., orally; through injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraarterial, intraportal, or intralesional routes; by sustained release systems (which may also be injected); or by implantation devices. Where desired, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device.

Alternatively or additionally, the composition can be administered locally via implantation of a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration.

Therapeutic Uses of Dual Function Proteins

Proteins of the invention can be used to treat, diagnose, ameliorate, or prevent a number of diseases, disorders, or conditions, including, but not limited to metabolic disorders. In one embodiment, the metabolic disorder to be treated is diabetes, e.g., type 2 diabetes mellitus. In another embodiment, the metabolic disorder is obesity. Other embodiments include metabolic conditions or disorders such as type 1 diabetes mellitus, pancreatitis, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, hypertension, cardiovascular disease, acute myocardial infarction, atherosclerosis, peripheral arterial disease, stroke, heart failure, coronary heart disease, kidney disease, diabetic complications, neuropathy, disorders associated with severe inactivating mutations in the insulin receptor, lipodystrophies including HIV-associated lipodystrophy, gastroparesis and other metabolic disorders.

In application, a disorder or condition such as type 1 or type 2 diabetes mellitus or obesity can be treated by administering a dual function protein variant as described herein to a patient in need thereof in the amount of a therapeutically effective dose. The administration can be performed as described herein, such as by IV injection, intraperitoneal injection, intramuscular injection, or orally in the form of a tablet or liquid formation. In most situations, a desired dosage can be determined by a clinician, as described herein, and can represent a therapeutically effective dose of the dual function protein polypeptide. It will be apparent to those of skill in the art that a therapeutically effective dose of dual function protein polypeptide will depend, inter alia, upon the administration schedule, the unit dose of antigen administered, whether the nucleic acid molecule or polypeptide is administered in combination with other therapeutic agents, the immune status and the health of the recipient. The term “therapeutically effective dose,” as used herein, means that amount of dual function protein polypeptide that elicits the biological or medicinal response in a tissue system, animal, or human being sought by a researcher, medical doctor, or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositions comprising one or more of the dual function proteins of the invention or mutants described herein and a pharmaceutically acceptable carrier. In some embodiments the pharmaceutical compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier. Pharmaceutically acceptable salts can also be present in the pharmaceutical composition, e.g., mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington: The Science and Practice of Pharmacy (1995) Alfonso Gennaro, Lippincott, Williams, & Wilkins.

Fusion Proteins and Peptidic Compounds

In another embodiment, the proteins of the present invention can be made into a fusion protein or peptidic compound derived from the dual function proteins of the invention amino acid sequences. Such fusion proteins and peptidic compounds can be made using standard techniques known in the art. For example, peptidic compounds can be made by chemical synthesis using standard peptide synthesis techniques and then introduced into cells by a variety of means known in the art for introducing peptides into cells (e.g., liposome and the like).

The in vivo half-life of the fusion protein or peptidic compounds of the invention can be improved by making peptide modifications, such as the addition of N-linked glycosylation sites into the dual function proteins of the invention, or conjugating dual function proteins of the invention to poly(ethylene glycol)(PEG; pegylation), e.g., via lysine-monopegylation or cysteine-monopegylation. Such techniques have proven to be beneficial in prolonging the half-life of therapeutic protein drugs. It is expected that pegylation of the proteins of the invention of the invention may result in similar pharmaceutical advantages.

In addition, PEGylation can be achieved in any part of a polypeptide of the invention by the introduction of a non-natural amino acid. Certain non-natural amino acids can be introduced by the technology described in Deiters et al., J Am Chem Soc 125:11782-11783, 2003; Wang and Schultz, Science 301:964-967, 2003; Wang et al., Science 292:498-500, 2001; Zhang et al., Science 303:371-373, 2004 or in U.S. Pat. No. 7,083,970. Briefly, some of these expression systems involve site-directed mutagenesis to introduce a nonsense codon, such as an amber TAG, into the open reading frame encoding a polypeptide of the invention. Such expression vectors are then introduced into a host that can utilize a tRNA specific for the introduced nonsense codon and charged with the non-natural amino acid of choice. Particular non-natural amino acids that are beneficial for purpose of conjugating moieties to the polypeptides of the invention include those with acetylene and azido side chains. The proteins of the invention containing these novel amino acids can then be PEGylated at these chosen sites in the protein.

Similarly PEGylation can be achieved in any part of a protein of the invention by the introduction of pyrrolysine or pyrroline-carboxy-lysine as described by Ou et al. (Proc Natl Acad Sci USA. 2011 Jun. 28; 108(26):10437-42. Epub 2011 Jun. 13).

EXAMPLES Example 1: Design of GLP-1/Proteins of the Invention

In order to test the efficacy of GLP-1 and FGF21 together, a fusion molecule was designed. Because GLP-1 requires a free N-terminus and FGF21 requires a free C-terminus for receptor binding and activation, the two were cloned in the order of N-GLP-1-linker-FGF21-C. Initial constructs were made with GLP-1 residues 7-35 and with or without several modifications to the N-terminus for DPP-4 protection (discussed below). Further modifications of GLP-1 (point mutants or deletions) were tested and scored by in vitro potency.

Linkers of 3, 8, 10, or 20 amino acids were cloned. For FGF21, residues 33-209 of the wild-type human protein were used. Constructs with and without PEGylation sites were produced. PEGylation was accomplished using maleimide-PEG reagents (40 kDa linear and branched PEGs) reactive toward an introduced Cys. The Cys was placed at position R154 of FGF21 and at several sites in the GLP-1 or Exendin-4 and linker. Constructs have also been designed for modification through incorporation of a Pcl, Pyl, Pyl analog or a reactive non-naturally occurring amino acid by introducing a TAG (amber) codon at position R154 of FGF21 or K34 of GLP-1. Additional constructs were designed for two different modifications by inclusion of the Cys at R154 or K34 and a TAG codon for incorporation of a Pcl, Pyl, Pyl analog or a reactive non-naturally occurring amino acid at the other site. Constructs were made fusing GLP-1 and FGF21 to an Fc domain of a human antibody. Variant 76 (V76) sequence of FGF21 was introduced into the fusion format.

Expression and purification of the fusions: Fusion proteins were typically expressed in E. coli by cloning the DNA sequence encoding the particular dual function variant into a vector containing coding sequences for one of several removable domains (for example, NPro or NPro variants with or without His6 tag, or His6 tag with a TEV-protease recognition peptide, or His6-Ubiquitin, or His6-Smt3) The vectors further included an inducible promoter to initiate mRNA transcription for protein expression such as the lac, T7 or arabinose promoters). Briefly, the vectors were transformed to DH10b-derived E. coli cells or BL21(DE3)-derived cells, grown under standard conditions, and induced to express protein with 0.2% arabinose or IPTG added to the culture media. Cells were harvested 3-4 hours post-induction, spun, and frozen. Pellets were thawed and resuspended for lysis by sonication and insoluble protein was isolated by centrifugation. The pellet was then solubilized in 6M guanidine, and the dissolved, clarified proteins were loaded to Ni-NTA columns. The columns were washed with denaturing and then non-denaturing buffers and eluted in a non-denaturing buffer according to standard practice. The eluates were buffer exchanged to remove imidazole, digested with specific proteases for removal of tags, (for example TEV protease, ubquitinase Usp2, or Ulp1 for specific removal of smt3), and purified by Ni-NTA. The fusion protein-containing fractions were further purified by size exclusion chromatography before PEGylation (typically with NOF Sunbright GL2-400 MA 40 kDa branched PEG maleimide). PEGylated proteins were isolated by anion exchange chromatography, placed in PBS, and concentrated or stored for various assays.

Protection of GLP-1 by mutation of the N-terminus: Because processing of the N-terminus of GLP-1 by DPP-4 is known to inactivate the peptide toward its primary receptor, several mutations reported in the literature were explored to slow this process. Constructs with the addition of an extra glycine to the N-terminus, mutation of Glu9 to Pro, or mutation of Ala8 to Gly or Ser, were all tested in a cell-based assay of GLP-1 activity. In the context of the fusion to FGF21 with a 40 kDa PEG attached to FGF21, the mutations at Ala8 retained superior activity to the other N-terminal modifications (FIG. 1a ), and the A8S mutation was used in subsequent designs. In addition, the DPP-4-resistant analogue, Exendin-4 was also tested in the fusions as an alternative moiety with extended half-life in vivo (FIG. 1b ). [See, e.g., J. Y. Oh et al. (2009) Bulletin of the Korean Chemical Society 30, 2471-2474; K. Adelhorst, (1994) JBC 269, 6275; B. D. Green et al. (2003) Biological Chemistry 384, 1543; J. C. Parker et al. (1998) Journal of Peptide Research 52, 398; C. F. Deacon et al. (1998) Diabetologia 41, 271; R. Burcelin et al. (1999) Metabolism-Clinical and Experimental 48, 252; U. Ritzel et al. (1998) Journal of Endocrinology 159, 93.]

The A8S mutation was also compared to wild-type GLP-1 for pharmacokinetic properties in vivo. A fusion protein with wild-type GLP-1 (V329) and one with the GLP-1(A8S) mutation (V232) were injected intravenously into rats at 1 mg/kg, and the serum levels were measured against standard curves produced with the dosing solutions. When measured with a human FGF21-reactive ELISA kit, the molecules appeared to behave similarly to each other and to FGF21 alone with the same size (40 kDa) PEG attached (FIG. 2). To measure the half-life extension of GLP-1 activity, C57BL/6J mice were injected with the same two dual function protein variants with either wild-type GLP-1 or GLP-1(A8S). While both acutely lowered the fed glucose level after dosing, the GLP-1(A8S) variant retained greater activity in an oral glucose tolerance test (OGTT) measurement 3 days after dosing (FIG. 3). These data demonstrate that the A8S mutation increases the effective half-life of the GLP-1 moiety of the fusion while the PK of the FGF21 moiety is not much affected by the addition of GLP-1.

Design of the peptide linker between GLP-1 and FGF21: Linkers of 3, 8, 10, or 20 amino acids were tested. No significant difference in activity was observed for the variants with different linkers, although there were differences in expression yields in some fusion contexts. Additionally, constructs were tested with various lengths of GLP-1 (8 to 31 residues) or Exendin-4 (30 or more residues) and tested for in vitro activity in the GLP-1R cell-based assay.

Example 2: Test of Fusions in the Ob/Ob and Db/Db Diabetic Mouse Models

FGF21 has been shown to improve blood glucose levels, liver lipid levels, and body weight in the ob/ob mouse model of type-2 diabetes (T2D; see, e.g., A. Kharitonenkov et al. (2005) Journal of Clinical Investigation 115, 1627; T. Coskun et al. (2008) Endocrinology 149, 6018; and E. D. Berglund et al. (2009) Endocrinology 150, 4084). Likewise, GLP-1 analogues have been shown to improve glucose control, beta cell function and liver health in this genetic mouse diabetes model (Gallwitz B., Glucagon-like peptide-1 as a treatment option for type 2 diabetes and its role in restoring beta-cell mass. Diabetes Technol Ther. 2005, 7:651-7).

To determine if the fusion of GLP-1 to FGF21 would lead to additional efficacy, the WT GLP-1 and GLP-1(A8S) versions were tested against an equivalent FGF21 molecule each with the same 40 kDa branched PEG attached. In a two week study with twice weekly dosing, the FGF21(R154C)-PEG (V238) and GLP-1-FGF21(R154C)-PEG (V239) showed similar effects on blood glucose, body weight, and liver health (assessed by alanine aminotransferase (ALT) serum levels, weight, and appearance) (FIG. 4). Glucose measurements showed a faster improvement for the fusion than for FGF21 alone after the first dose. The two converged at time points more than a day after dosing, suggesting that the additional benefit due to GLP-1 was shorter lived than the FGF21 efficacy.

The GLP-1(A8S)-FGF21(R154C)-PEG (V235) fusion also showed similar effects at 0.2 mg/kg, rather than the 1 mg/kg used for the other compounds. This compound also generally gave more consistent results between dosing, suggesting that the DPP-4-resistance was critical to the overall improvement in efficacy as compared to the up-and-down behavior of GLP-1-FGF21(R154C)-PEG (V239). It was also observed that the GLP-1(A8S)-FGF21(R154C)-PEG (V235) at 1 mg/kg showed additional lowering of glucose, body weight, ALT/AST, and liver weight.

Additional studies were conducted to see if the improved efficacy could be replicated with co-administration of separate FGF21 and GLP-1 compounds. To make dosing of proteins similar between groups, a tool compound was made in which 14 amino acids were removed from the FGF21 C-terminus. This molecule (V253) was at least 1000× less potent in the FGF21 receptor assay but retained equal potency in the GLP-1R assay as compared to the other fusions and was unable to inhibit wild-type FGF21 activity. This compound had pharmacokinetics in rat similar to the other fusions when assessed by FGF21 levels (FIG. 2).

Results of a four week study in which ob/ob mice were treated with FGF21(V76)-PEG, GLP-1(A8S)-PEG (V253), both together, or GLP-1(A8S)-FGF21(V76)-PEG fusion (V272) are shown in FIG. 5. The fusion was significantly more efficacious than co-treatment of the individual FGF21(V76)-PEG and GLP-1(A8S)-PEG molecules on fed glucose AUC, OGTT glucose AUC, body weight at the end of the study, hepatic lipid content, and ALT measurement (p-values <0.05 for all).

Serum exposure of human FGF21 and GLP-1 were checked in the terminal serum samples to confirm exposure in the various groups. Human FGF21 was detected in all treated groups, and a significant increase in active GLP-1 was measured in all groups treated with GLP-1 (A8S)-PEG (V253) or GLP-1(A8S)-FGF21(V76)-PEG (V272). The GLP-1(A8S)-FGF21(V76)-PEG (V272) group showed similar levels of human FGF21 to the 0.2 mg/kg FGF21(V76)-PEG group, suggesting that the improved efficacy is not due to a higher systemic accumulation of the fusion in the animals. The GLP-1(A8S)-PEG (V253) molecule was fully active in vitro (Table 8). These data demonstrate that the efficacy of the fusion was dependent on both moieties being in a single molecule, and, e.g., benefitted from a synergy at the cellular level.

To further test GLP-1(A8S)-FGF21(V76)-PEG (V272) vs. co-administration, three studies were conducted in db/db mice. In each study, male db/db mice (8-11 weeks old were dosed i.p. twice weekly. In an initial three week study, the dual function fusion protein, GLP-1(A8S)-FGF21(V76)-PEG (V272), matched the glucose lowering of the co-administration at equal doses and surpassed the efficacy of single entity dosing. The dual function fusion protein also showed superior body weight loss.

In a second two week study, GLP-1(A8S)-FGF21(V76)-PEG (V272) showed similar glucose lowering compared to FGF21(V76)-PEG at a 25-fold lower dose and GLP-1 (A8S)-PEG (V253) at a 5-fold lower dosing. In addition, GLP-1(A8S)-FGF21(V76)-PEG (V272) lowered HbA1c significantly more and lowered body weight significantly more than the mono-therapies or the maximally efficacious dose of the co-administration groups (FIG. 8, Table 2). Unexpectedly, altering the dosing ratio of the co-treatments higher than 1:1 did not improve the observed efficacy (Table 2). This suggests that dual function proteins including equal numbers of GLP-1 moieties and FGF21 moieties can achieve optimal efficacy during in vivo treatments.

TABLE 2 Therapeutic Dosages Dose HbA1c, Body weight, (mg/kg change from day 12 Treatment 2x/wk) initial (%) (g) Vehicle N/A  1.6 ± 0.9 42.3 ± 2.1 V76 1  2.2 ± 1.0 43.3 ± 2.1 V76 3  1.0 ± 0.9 42.3 ± 1.3 V76 5  0.7 ± 0.4¹ 42.1 ± 1.9 V253 1  0.8 ± 0.7¹ 42.6 ± 2.1 V253 3  0.6 ± 0.8¹ 40.9 ± 2.6 V253 5 −0.1 ± 0.6¹ 40.4 ± 2.1 V76 + V253 1 + 1 −0.3 ± 0.6¹ 37.7 ± 2.2¹ V76 + V253 1 + 3  0.5 ± 0.6¹ 37.7 ± 1.7¹ V76 + V253 3 + 1 −0.1 ± 0.5¹ 38.9 ± 1.2¹ V76 + V253 3 + 3 −0.3 ± 0.4¹ 36.0 ± 1.7¹ V272 0.2  0.4 ± 1.1¹ 41.1 ± 2.7 V272 1 −1.1 ± 0.5^(1,2) 33.8 ± 2.1^(1,2) ¹p-value vs. vehicle <0.05. ²p-value vs. co-treatment groups <0.05

Table 2 shows efficacy of mono-therapies and co-treatments at different doses compared to dual function proteins in a db/db mouse study with 2 weeks of dosing. The reported values are averages of measurements made in eight animals with standard deviation. In a four week study, male db/db mice (n=8) were dosed twice weekly for four weeks with mono-therapies, co-treatments, or dual function fusion proteins. On day 27, the mice were fasted and the blood insulin levels were measured for all animals. At the end of the study, pancreatic insulin contents were extracted and measured (n=3). GLP-1(A8S)-FGF21(V76)-PEG (V272) showed a robust dose response from 0.1-1 mg/kg with efficacy equal to or better than FGF21(V76)-PEG or GLP-1 (A8S)-PEG (V253) alone on glucose levels, HbA1c, and body weight at more than 10-fold lower dose, and improved efficacy over the maximally efficacious dosing of the co-administration. GLP-1(A8S)-FGF21(V76)-PEG (V272) also lowered fasting insulin levels in the serum while increasing insulin content of the pancreas to a greater extent than the single or combination treatments of GLP-1(A8S)-PEG (V253) and FGF21(V76)-PEG (FIG. 7). GLP-1(A8S)-FGF21(V76)-PEG (V272)-treated animals were close to lean control mice in fed glucose, HbA1c, and body weight numbers.

To estimate metabolic rate and substrate utilization, we measured oxygen consumption and carbon dioxide production using an Oxymax indirect calorimetry system (Columbus Instruments, Columbus, Ohio). Mice were housed in the chamber with a 12-h light/12-h dark cycle in an ambient temperature of 22-24° C. VO₂ and VCO₂ rates were determined under Oxymax system settings and protocol. The system was calibrated against a standard gas mixture to measure O₂ consumed (VO₂, ml/kg/h) and CO₂ generated (VCO₂, ml/kg/h). Metabolic rate (VO₂) and respiratory exchange ratio (RER) (ratio of VCO₂/VO₂) were evaluated over a 72-h period. Calculated RER values indicated that animals treated with fusion protein relied more on lipid substrates (RER values close to 0.75-0.85) particularly during the 24-72 hours after the second dose when compared to vehicle or combination treated groups. During this period, vehicle and combination treated groups showed RER values were close to 0.9 suggesting more of carbohydrate substrate utilization for energy and to a lesser extent lipid substrate utilization for energy expenditure. The data suggest that fusion protein treatment caused an increase in fatty acid oxidation that may be contributing to its body weight reduction effect in db/db mice

A dual function fusion protein, when compared to combination, showed improved beta-cell function in vivo. After four doses of either dual function protein GLP-1(A8S)-FGF21(V76)-PEG (V272) or GLP-1(A8S)-PEG (V253) plus FGF21(V76)-PEG, db/db mice were dosed orally with glucose and arginine. The glucose excursion of the dual function protein-treated group (72% lower than vehicle) was significantly lower than the combination-treated group (56% lower than vehicle). During the experiment, the amount of insulin secreted was also higher for the dual function protein-treated group (279% higher than vehicle) than for the combination-treated group (122% higher than vehicle).

Overall, the figures and tables, as well as data not shown herein, demonstrate the ability of the dual function fusion proteins of the invention, e.g., GLP-1(A8S)-FGF21(V76)-PEG (V272), to improve metabolic parameters for in vitro and in vivo rodent models of diabetes, when compared to a combination of individual GLP-1 and FGF21 (e.g., FGF21(V76)-PEG and GLP-1(A8S)-PEG (V253)). Said improved parameters (as used herein, “metabolic parameters”) include but are not limited to fed glucose (AUC), body weight, liver triglycerides, plasma HbA1c, serum triglyceride levels total cholesterol levels, oral glucose tolerance test serum glucose measurement (AUC), fasting serum insulin, pancreatic insulin content, and body fat percentage.

Example 3: Design of GLP-1/Proteins of the Invention from FGF21 Variants

A fusion was made with the following FGF21 variant sequence, which we refer to herein as “Variant 76” or “V76”:

(SEQ ID NO: 130)   1 DSSPLLQFGG QVRQRYLYTD DAQETEAHLE IREDGTVGGA AHQSPESLLE LKALKPGVIQ  61 ILGVKTSRFL CQKPDGALYG SLHFDPEACS FRELLLEDGY NVYQSEAHGL PLHLPGNRSP 121 HCDPAPQGPA RFLPLPGLPP ALPEPPGILA PQPPDVGSSD PLAMVGPSQG RSPSYAS

FGF21(V76)-PEG features a 40 kDa branched PEG linked through Cys154, and eight point mutations relative to the 177 amino acid wild-type protein (Q56E, D74H, Q82E, R105K, K150R, R154C, R159Q, S195A, all made relative to full-length FGF21 protein sequence (NCBI reference sequence number NP_061986.1)). The risk of clinical immunogenicity toward this molecule is considered low based on an EpiScreen time course assay of human T-cell responses. The molecule has a serum half-life time of more than 30 h in mouse and rat and significantly lowers glucose AUC in ob/ob mice with twice-weekly injections of 1 mg/kg.

The GLP-1(A8S)-FGF21(V76)-PEG (V272) molecule was tested against the GLP-1(A8S)-FGF21(R154C)-PEG (V235) fusion at three doses (0.05, 0.1 and 0.2 mg/kg). Both fusions showed a dose response in lowering glucose AUC, body weight, food intake, and ALT (FIG. 6). GLP-1(A8S)-FGF21(V76)-PEG (V272) was generally equal or better and showed higher efficacy for all parameters at the 0.2 mg/kg dose. GLP-1(A8S)-FGF21(V76)-PEG (V272) also showed lowering of serum triglycerides and cholesterol at both 0.1 and 0.2 mg/kg. Based on these data, the GLP-1(A8S)-FGF21(V76)-PEG (V272) molecule shows similar or improved properties to the initial fusions and is suitable for further study and development.

Ex4(1-30)-L20-FGF21(V76)-PEG (V277) also showed similar efficacy to GLP-1(A8S)-FGF21(V76)-PEG (V272) in an ob/ob 2 week study for glucose control, body weight and lipid levels.

Choice of PEGylation site for half-life extension. As seen for example in FIG. 2, PEGylation was used to extend the half-life of the molecule from a few minutes for GLP-1 or less than an hour for FGF21 to longer than 30 hours as measured in rats. To see if the two moieties of the fusion could be modulated by placement of the PEG, a series of constructs was made with the extra cysteine not in the FGF21 sequence but in that of GLP-1 or the linker. These constructs were tested for both GLP-1 and FGF21 activity in cell-based assays. Experiments reveal a stretch of positions within the fusion protein sequences at which PEGylation does not greatly impact either activity. Careful placement of the PEG at the N-terminal end of this stretch could be used to make a molecule with knocked-down GLP-1 activity while placement in the linker could be used to knock-down FGF21 activity. Such a molecule may be useful in tuning the potency of the fusion (for example, if the high potency of GLP-1 or Exendin-4 resulted in a poor therapeutic window when dosed at the efficacious level of a lower potency FGF21).

Based on these data, fusions with PEGylation at K34C of GLP-1 (V273) or K27C of Exendin-4 (V274) were prepared with FGF21(V76-C154R) whose sequence is as follows:

(SEQ ID NO: 131) DSSPLLQFGG QVRQRYLYTD DAQETEAHLE IREDGTVGGA  AHQSPESLLE LKALKPGVIQ ILGVKTSRFL CQKPDGALYG  SLHFDPEACS FRELLLEEGY NVYQSEAHGL PLHLPGNRSP HRDPAPQGPA RFLPLPGLPP ALPEPPGILA PQPPDVGSSD  PLAMVGPSQG RSPSYAS

A two week study in ob/ob mice demonstrates that, although the new fusions (V273 and V274) exhibited potency in vitro similar to the previous fusions and similar pharmacokinetics in rat, they were not as efficacious in vivo. At 0.2 mg/kg, the GLP-1 fusion variant showed no significant efficacy for glucose or body weight. The Exendin-4 fusion variant showed significant lowering of ALT and body weight and a trend toward lower AST compared to vehicle but no significant effect on blood glucose. Although these fusions may show efficacy at higher doses, the original format with PEGylation at R154C in FGF21 was chosen for further study. It is unclear if this unexpected difference indicates a special property of the GLP-1(A8S)-FGF21(V76-154R)-PEG version of the fusion that is blocked by PEGylation at the alternate site or if there is another explanation such as the PEG blocking the GLP-1R interaction more on the native cells with native receptor levels than on the transfected cells used for the in vitro assay.

Example 4: Further Characterization of GLP-1/FGF21 Proteins of the Invention

In order to further optimize and rank the GLP-1-FGF21-PEG dual activity proteins of the invention, and to gain a fuller appreciation of their improved efficacy over co-administration models, the fusions are tested in the ob/ob model of efficacy. These include but are not limited to molecules with longer or shorter linkers, additional proteins of the invention (which may include but are not limited to mutants based on Variant 76 or additional variants in development such as additional or different point mutations, insertions or deletions, dimerized molecules, alternative PEGylation strategies, molecules with additional disulfide bridges, molecules produced in different expression systems), and variants of Exendin-4 or GLP-1 to improve serum stability or activity. Candidates are also filtered by in silico immunogenicity prediction, expression levels, and quality of final products (related to solubility, aggregation and stability).

In order to further characterize the synergy of FGF21 and GLP-1 for treatment of diabetes and obesity, interaction dynamics of the two molecules are mapped, and the fusion proteins are compared to the maximally efficacious co-administration of the individual parent molecules. These experiments are conducted in models that focus on obesity and weight loss (Diet Induced Obesity in rat or mouse) and those that better model aspects of diabetes such as hyperglycemia, dislipidemia, and impaired beta cell function (ob/ob or db/db mice). The extent of synergy achieved by the dual activity proteins of the invention can be further determined by mapping the efficacy of each individual molecule, the combination of the two at different ratios, and the fusion.

Testing in cell-based assays with primary or immortalized rat islets and primary human islets is conducted to assess the efficacy of the fusion for cell proliferation, protection from apoptosis, and function, e.g., to validate the dual activity proteins of the invention in accepted models of Type 1 Diabetes (see further Van Belle, T. L. et al. Drug Discovery today: disease models. 209 pp. 41-45). Preferred in vivo models are those featuring pancreatic ablation by streptozotocin (STZ); targeted beta-cell ablation in a genetically modified and inducible mouse strain (RIP-DTA) in which the beta-cells are destroyed by supplementing the diet with doxycycline to induce targeted expression of diphtheria toxin; or the non-obese diabetic (NOD) mouse model of Type-1 diabetes with an autoimmunity mechanism. In these models, testing is possible in both prophylactic dosing to assess beta-cell protection (particularly in NOD) as well as post-challenge dosing to assess beta-cell stimulation and proliferation (particularly in STZ and RIP-DTA).

The following experiments are conducted to more fully realize the mechanism of efficacy synergy achieved by the dual activity proteins of the invention over the co-administration regimens: Cells co-transfected with both receptors (GLP-1R; FGFR1(IIIc), 2(IIIc), 3(IIIc), or 4; and beta-klotho) can be used to determine if the receptors are able to potentiate the opposing signal at the cell surface. Crosstalk between downstream signals can be detected in cells naturally expressing both receptors (e.g., beta cells). In animals, the contribution of food intake (investigated with pair-fed cohorts), increased metabolic rate (investigated in clamp and metabolic chamber experiments), and other mechanisms could be more thoroughly investigated to elucidate a mode of action for the synergistic effects of the two signals. Gene expression profiling of key tissues (liver, pancreas, adipose, intestine, heart, aorta, brain, etc.) may also be conducted in order to elucidate the unique signaling of the fusion proteins, potentially accounting for their improved efficacy.

The following experiments are conducted to understand the activity of dual function proteins of the invention to control or modify differentiation of mesenchymal stem cells (MSC): MSC can be treated with proteins of the invention alone or with compounds or mixtures of compounds known to induce osteogenesis or with compounds or mixtures of compounds known to induce adipogenesis and the resulting rates of differentiation to osteoblast-like cells or adipocyte-like cells can be measured.

Example 5: Design of GLP-1 and Exendin-4-Containing Proteins of the Invention from Fc-Fusion FGF21 Variants

Constructs were made with alternative linkers (for example, SGGGGSGGGGSGGGGSA (SEQ ID NO:138), GGGGS (SEQ ID NO:173), GG, and GGGGSGGGGSGGGGS (SEQ ID NO:174)), FGF21 variants, and GLP-1-related peptides. Typically the dual function proteins containing an Fc domain were expressed in HEK293 cells. The DNA coding sequences of these fusions were cloned into vectors containg sequence encoding a leader peptide to direct the proteins for secretion and further containing sequences necessary to promote mRNA syntehsis and protein expression of the desired products. HEK293 cells were transiently transfected with the vectors. The media from these cell cultures were collected, filtered, and purified by protein A affinity chromatography. The eluates were brought to netural pH and further purified by size exclusion chromatography. The products were then concentrated and stored for various assays. Variants were tested for activity in vitro in four assays: CRE-luciferase expression induction in HEK293 transfected with GLP-1R (Table 3) and glucose stimulated insulin secretion in INS1E cells (Table 6) to measure activity through GLP-1R; ERK phosphorylation induction in HEK293 transfected with beta-klotho (Table 4) and 2-deoxyglucose uptake by 3T3L1 cells (Table 5) to measure FGF21 activity. Variants with GLP-1(A8S) and a 15 amino acid linker were more active than variants with GLP-1 and shorter linkers. Exendin-4 (1-39) variants with all linkers tested were active. Exendin-4 (1-30)-containing variants had similar in vitro potency to Exendin-4 (1-39)-containing constructs. A variant with a tandem repeat of Exendin-4(1-39) was not as potent as variants with a single copy of a GLP-1-related peptide.

TABLE 3 GLP-1R activation in HEK293-GLP-1R-CRE-Luciferase Reporter Cells. Variant# GLP-1R EC₅₀ (nM) V193 0.024 V194 0.042 V195 0.041 V196 0.027 V197 0.085 V198 0.024 V199 0.046 V202 0.025 V203 0.252 V206 0.047 V207 0.088 V208 0.044 V209 0.033 V210 0.038 V211 0.032 V212 5.90 V213 18.4 V214 0.023 V215 8.99 V216 0.025 V217 4.53

TABLE 4 Activity measured in an ERK phosphorylation assay of FGF21 activity in HEK293-KLB cells. βklotho pERK % γ-max vs. Potency Variant# EC₅₀ (nM) WT FGF21 (Fold over WT FGF21) V193 2.1 101 1.2 V194 9.8 97 5.8 V195 13 76 7.6 V196 0.87 58 0.18 V197 0.92 63 0.19 V198 0.94 67 0.24 V199 1.04 58 0.27 V202 1.78 67 0.37 V203 1.01 70 0.21 V206 0.51 83 0.15 V207 1.10 83 0.34 V208 0.53 54 0.20 V209 0.70 83 0.21 V210 0.84 88 0.24 V211 0.58 73 0.17 V212 1.24 89 0.37 V213 1.00 61 0.37 V214 0.55 50 0.21 V215 0.92 51 0.34 V216 0.85 56 0.32 V217 2.23 46 0.83

TABLE 5 Activity measured in a 2-deoxyglucose uptake in 3T3L1 mouse adipocytes. 2DOG % γ-max vs. Potency Variant# EC₅₀ (nM) WT FGF21 (Fold over WT FGF21) V194 2.8 118 1.6 V196 0.055 90 0.14 V197 0.077 100 0.18 V198 0.079 105 0.21 V199 0.090 101 0.37 V202 0.45 86 2.5 V203 0.078 100 0.31 V206 0.093 85 0.34 V208 0.067 87 0.25 V209 0.157 91 0.59 V210 0.067 98 0.27 V211 0.073 99 0.29 V214 0.213 87 0.81 V216 0.137 80 0.56

TABLE 6 Activity measured by glucose-stimulated insulin secretion (GSIS) in INS1E rat insulinoma cells. GSIS EC50 (nM) Variant# (avg of 2) V196 9.1 V197 46 V198 4.4 V199 5.0 V202 33 V203 128 V206 2.4 V208 1.6 V209 1.9 V210 2.7 V211 1.5 V214 4.6 V216 6.8

Eight ob/ob mice were dosed with variants twice per week at 0.5 mg/kg for two weeks. Blood glucose and body weight were measured during the studies, and hepatic lipids were measured at the end of the study. As seen in the following table (Table 7), treated animals had reduced blood glucose, lower body weight, and lower hepatic lipids throughout the study, when compared to vehicle-treated age-matched animals.

TABLE 7 Summary of results of treating ob/ob mice twice per week at 0.5 mg/kg for two weeks. Fed AUC TG FASTED Hepatic BW loss, D12 lowering (8 h, or O/N) Lipids Variant# (% of Veh) (% of Veh) (% of Veh) (% of Veh) V193 −20* −55* 39 −66* V195 −7* −30* N/A −10  V196 −23* −63*  8 −65* V197 −16* −53* 52 −85* V198 −24* −64* −7 −69* V199 −24* −64* −7 −69* V200 −14* −39* −5 −62* V201 −22* −60* −10  −58* V202 −18* −59* 22 −63* V203 −8* −33*  68* −73* V206 −16* −58* −19  −56* V207 −22* −64* −10  −55* V208 −21* −60* 10 −57* V209 −28* −66* −11  −62* V210 −23* −67* −14  −57* V211 −28* −68*  0 −52* V214 −19* −58* 110* −41* V216 −21* −62* 29 −51* *p-value vs. vehicle <0.05

Example 6: Further Characterization of Proteins of the Invention in Receptor Pharmacology Assays

As seen, for example, in FIG. 9, dual agonist proteins of the invention were compared to various forms of FGF21 and Exenatide for their ability to induce ERK phosphorylation in HEK293 transfected with beta-klotho. At 10 minutes, cells treated with GLP-1(A8S)-FGF21(V76)-PEG (V272) showed a higher potency and higher maximal proportion of phospho-ERK to total ERK when compared to cells treated with FGF21(V76)-PEG plus Exenatide. The higher maximal phospho-ERK signal shows that the dual function protein V272 is a superior agonist for signaling through FGFR1c/beta-klotho when compared to the FGF21(V76)-PEG molecule, possibly due to better interaction with the receptor (for example, the N-terminal extension causing the dual function protein to attain a more favorable structure for receptor binding or activation).

Select dual function proteins were tested for their ability to induce phospho-ERK signaling in human adipocytes. When compared to a single function FGF21 molecule (V101), the dual function proteins V208, V209, V211, V14, and V272 were more potent for stimulating phosphorylation of ERK. Because these are human cells, it may also suggest that the dual function proteins of the invention will be highly active in treatment of human disease when compared to treatments including other FGF21 mimetics, similar to what has been demonstrated for activity in rodents to date.

As seen, for example, in Table 8 and FIG. 10, dual agonist proteins of the invention were compared to Exenatide, GLP-1 peptide, Exendin-4(1-39)-L5-Fc (V201), and GLP-1(A8S)-PEG (V253) for their ability to signal through GLP-1R in HEK293 cells transfected with GLP-1R or cells co-transfected with GLP-1R, beta-klotho and FGFR1c. Cells were treated with compound for 30 minutes, and cAMP levels were measured. Table 8 shows that in cells with both the GLP-1R and the FGF21 receptor complex, the dual agonist proteins (V272 and V211) showed higher potency than the single agonists (V253 and V101) alone or in combination with Exendin-4. In cells with only GLP-1R, the potency of the single agonists alone or in combination with Exendin-4 was equal to that of the dual agonist proteins. FGF21 variants alone were inactive in the assay.

TABLE 8 Assay of cAMP induction in cells treated with dual function proteins of the invention. GLP-1R/FGFR1c/beta- klotho GLP-1R only EC50 y-max EC50 y-max EC50 (fold- (% Ex- EC50 (fold-Ex- (% Ex- Compound (nM) Ex-4) 4) (nM) 4) 4) Ex-4 6.0 1.0 100 11 1.0 100 V253 24 4.1 92 34 3.2 81 V253 + V76 12 2.1 91 38 3.5 82 V272 2.4 0.4 97 64 6.0 76 V201 26 4.4 93 32 3.0 100 V201 + V101 15 2.4 98 18 1.7 82 V211 6.6 1.1 92 39 3.6 98

Two additional HEK293 cell lines were generated in Discoverx's PathHunder assay format which utilizes complementation of beta-galactosidase fragments on beta-arrestin and the C-terminus of GLP-1R to measure recruitment of arrestin upon receptor activation. FIGS. 10a-10d show that with a 1 hour incubation of cells with protein or peptide, GLP-1 (A8S)-FGF21(V76)-PEG (V272), Exendin-4(1-39)-L15-Fc-L15-FGF21(V103) (V211), GLP-1(A8S)-PEG (V253), and Exendin-4(1-39)-L5-Fc (V201), were less potent than Exenatide for recruitment of beta-arrestin and that no difference in behavior was noted whether cells contained GLP-1R/FGFR1c/beta-klotho or GLP-1R alone.

The receptor pharmacology studies presented here suggest that the dual function proteins of the invention will be superior in activity and efficacy when compared to combinations or co-treatments of conventional FGF21 and GLP-1/Exenatide molecules. The PEGylated dual function protein GLP-1(A8S)-FGF21(V76)-PEG (V272) unexpectedly showed higher potency and maximal signal for phospho-ERK induction when compared to the FGF21(V76)-PEG molecule. These data show that the dual function proteins are equivalent or superior for FGF21 signaling when compared to other FGF21 variants. Likewise, the dual function proteins exhibit higher potency for cAMP signaling downstream of GLP-1R when cells express both the GLP-1 and FGF21 receptors. These properties may reflect a direct interaction between the receptors or an indirect effect such as a boost in local concentration through avidity toward the two receptors. These observations suggest a mechanism for improved activity of the dual function proteins that may explain why co-treatments of individual GLP-1 and FGF21 variants have not been sufficient to match the in vivo efficacy of the dual function proteins, as presented in other examples of this invention.

Example 7: Characterization of Proteins of the Invention in Models of Type-1 Diabetes (T1D)

RIP-DTA transgenic mice (9-10 weeks old) were treated with doxycycline for 3 days to induce controlled beta-cell ablation (similar to Thorel et al., Nature 2010(464)1149-1154). The mice were then treated for three weeks, twice per week with FGF21(V76)-PEG, FGF21(V76)-PEG+GLP-1(A8S)-PEG (V253), or GLP-1(A8S)-FGF21(V76)-PEG (V272). Treated animals in all three groups showed lower basal fed glucose levels during the study. The dual function protein treatment resulted in significantly lower glucose AUC (64% reduced from vehicle) compared to combination treatment (55% reduced from vehicle). After fasting, the mice were given a glucose bolus, and glucose excursion was measured. All three treatments lowered blood glucose levels compared to vehicle-treated animals, with the dual function protein treatment resulting in a 76% reduction compared to 66% for the combination treatment. All treated groups had increased pancreatic insulin content compared to vehicle, with the dual function protein treatment resulting in a 76% greater insulin content than combination treatment.

NOD mice were treated twice per week for three weeks with GLP-1(A8S)-FGF21(154C)-PEG (V235) or GLP-1(A8S)-PEG (V253)+FGF21(154C)-PEG (V238). After two weeks, the mice were fasted and given a glucose bolus for an OGTT. The dual function protein-treated mice showed significantly lower fasted glucose (˜60% of vehicle) and glucose excursion during challenge (˜45%) compared to vehicle while combination-treated animals had fasted glucose levels that were not significantly different from vehicle (˜70%) and similar glucose excursion during challenge compared to vehicle-treated animals. At the end of the study, the dual function protein-treated animals had significantly lower basal glucagon levels (˜50%) than vehicle, while combination-treated animals had levels similar to vehicle.

Example 8. Predictive Immunogenicity Results—MHC-Associated Peptide Proteomics (MAPPs) and T-Cell Assay Results

The formation of anti-drug antibodies (ADA) to mAbs and other therapeutic proteins could potentially lead to severe immunotoxicological reactions, such as IgE-mediated anaphylactic reactions (Chung et al. (2008) N Eng J Med, 358, 1109-17) or immune complex disease, e.g., vasculitis, glomerulonephritis (Descotes and Gouraud (2008) Expert Opin Drug Metab Toxicol 4, 1537-49) as well as to a loss of clinical exposure and efficacy. Some patients treated with the therapeutic proteins PEGylated megakaryocyte growth and development factor (PEG-MGDF) and erythropoietin (EPO; Eprex) developed neutralizing ADA that were cross-reactive to their respective endogenous counterparts, leading to severe thrombocytopenia with PEG-MGDF and pure red-cell aplasia with Eprex (Li et al. (2007) Blood 98, 3241-8; Casadevall et al. (2002) N Engl J Med 346, 469-75). Hence, it is important to assess the immunogenicity risk prior to human testing. Evaluating the immunogenicity risk and putting in place a solid immunogenicity risk mitigation plan for the clinical development phase will be especially important for therapeutic proteins containing modified, non-human sequences such as the FGF21 variants that are the subject of this invention.

Formation of ADA can be induced in at least two different ways. T cell-dependent and -independent pathways have been described for B cell activation. A strong, high affinity IgG response is T cell-dependent and requires involvement of CD4+T helper cells (TH cells). The immune response is analogous to a response against foreign antigens: naive TH cells are specifically activated by professional antigen presenting cells (APCs), such as dendritic cells (DCs), and in turn induce activation of drug-specific B cells.

The MHC-associated Peptide Proteomics (MAPPs) assay involves in vitro identification of HLA class II associated peptides, which are processed by professional antigen presenting cells (APCs) such as dendritic cells. Antigen uptake, processing and presentation processes are taken into account. In this approach, immature human monocyte-derived DCs of different healthy blood donors, are incubated with different biotherapeutic drug candidates in the presence of an activation stimulus. The naturally processed HLA class II-associated peptides, which are derived from the biotherapeutic protein, are identified by liquid chromatography-mass spectrometry (Kropshofer and Spindeldreher (2005) in Antigen Presenting Cells: From Mechanisms to Drug Development, eds. Kropshofer and Vogt, Wiley-VCH, Weinheim, 159-98).

T cell assays provide a format in which the potential risk for immunogenicity of whole protein therapeutics can be assessed. T cell assays evaluate the capacity of a therapeutic protein to induce a CD4⁺ T cell response. Using a cohort of healthy blood donors covering a broad panel of HLA class II haplotypes, purified therapeutic proteins are tested for T cell proliferation and/or cytokine secretion in vitro. This technology been used successfully to compare protein variants for the potential to induce an immune response in vivo (Jones et al. (2004) J Interferon Cytokine Res. 24, 560-72.; Jones et al. (2005) J Thromb Haemost. 3, 991-1000) and this assessment of currently approved monoclonal antibodies does show some degree of correlation between the activation of T cells observed in vitro and immunogenicity in the clinic (Perry et al. (2008) Drugs R D 9, 385-96). In the context of this invention, peripheral blood mononuclear cells (PBMCs) from a cohort of 50 healthy donors representing the world population (based on HLA allotypes) are incubated with the FGF21 variants and T cell responses are measured using proliferation assays ([³]-Thymidine uptake) and IL-2 cytokine secretion (ELISpot). Subsequently, analysis of the frequency and magnitude of the CD4⁺ T cell responses are carried out to assess the risk of clinical immunogenicity.

The combined use of the T cell and MAPPs assays provides an effective process for evaluation of immunogenicity risk in the clinic using human cells. Modifications to a therapeutic protein candidate that results in a reduced number of peptides presented by dendritic cells and a reduced number of responding donors in the T-cell assay will be advantageous as these proteins bear a lower risk to develop immunogenicity in the clinic. An example of a protein modification that has been described to reduce immunogenicity is PEGylation. However, reduced immunogenicity does not always occur with PEGylation (Li et al. (2007) Blood 98, 3241-8).

In the MAPPs assay, all tested PEGylated GLP-1(A8S) and Exendin-4 FGF21-fusion molecules, including V272 (PEGylated) and V277 (PEGylated) and the non-PEGylated Fc-FGF21 variants containing the Q55C, G148C mutations (V101, V103 and V188), show a low number of clusters, and peptide length variants that are comparable to V76 (PEGylated) and lower than wild-type FGF21 or V76 (non-PEGylated). In the T cell assay, the frequency of T-cell responses was <10% of the study cohort and the magnitude of the responses were low for V272 (PEGylated) and V277 (PEGylated) and the non-PEGylated Fc-FGF21 variants containing the Q55C, G148C mutations (V101, V103 and V188) (Table 10). The absence of a PEG moiety or the absence of the additional disulfide bond at Q55C, G148C may contribute to the increased MAPPs and T-cell assay responses seen with wild-type FGF21 and V76 (non-PEGylated) (Tables 9 and 10). Based on the results from the MAPPs and T cell assays, the risk to develop immunogenicity in the clinic can be considered to be low for V76 (PEGylated), V272 (PEGylated), V277 (PEGylated) and the non-PEGylated Fc-FGF21 variants containing the Q55C, G148C mutations (V101, V103, V188). It is known that the addition of disulfide bonds to proteins can enhance their proteolytic stability and this feature of the Fc-FGF21 variants containing the Q55C, G148C mutations may contribute to the reduced number of peptides displayed by antigen-loaded mature dendritic cells in the MAPPs assay.

In addition, the increased MAPPs response in the assays to non-PEGylated V76 may be explained by the free cysteine leading to dimerization of the molecule and thereby impacting antigen processing and presentation. This is in line with the observation that wild type FGF21 with the introduced free cysteine showed the same tendency to dimerize and resulted in increased presentation of peptides. It is likely that additional PEGylated dual function proteins, and the GLP-1(A8S)-Fc-FGF21 variants (V202, V203, V212, V213, V214, V215, V216, V218 etc.) and the Exendin-4-Fc-FGF21 variants (V196, V197, V198, V199, V206, V207, V208, V209, V210, V211 etc.) containing the Q55C, G148C mutations, will also exhibit MAPPs and T cell assay responses that would be consistent with a low risk of developing immunogenicity in the clinic.

TABLE 9 T-cell assay summary with wild-type FGF21, V76 (non-PEGylated) and V76 (PEGylated) Experiment KLH Outcome T cell (positive FGF21-WT FGF21-WT- V76 (non- V76 assay control) (non-PEGylated) (PEGylated) PEGylated) (PEGylated) Proliferation % 90 6 4 16 4 ELISpot % 82 10 8 10 4 Proliferation 78 6 4 10 2 and ELISpot %

TABLE 10 T-cell assay summary with V76 (PEGylated), V272 (PEGylated), V277 (PEGylated), V101, V103 and V188 Experiment KLH Outcome T cell (positive V76 V272 V277 assay control) (PEGylated) (PEGylated) (PEGylated) V101 V103 V188 Proliferation % 92 4 6 4 4 6 4 ELISpot % 91 6 6 4 4 6 9 Proliferation 85 4 6 4 4 6 4 and ELISpot % 

What is claimed is:
 1. A method of treating a metabolic disorder by administering to a subject in need thereof a dual function fusion protein, wherein the dual function fusion protein comprises a GLP-1 receptor agonist, an FGF21 receptor agonist, and an Fc domain attached to each other via a GS linker, having an orientation of N-terminus-GLP-1 receptor agonist-linker-Fc domain-linker-FGF21 receptor agonist-C-terminus; and wherein the metabolic disorder is obesity, type 2 diabetes mellitus, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), insulin resistance, hyperinsulinemia, glucose intolerance, or hyperglycemia.
 2. The method of claim 1, wherein the dual function fusion protein comprises the amino acid sequence of SEQ ID NO:36.
 3. The method of claim 1, wherein the dual function fusion protein comprises the amino acid sequence of SEQ ID NO:134.
 4. The method of claim 1, wherein the dual function fusion protein comprises the amino acid sequence of SEQ ID NO:135.
 5. The method of claim 1, wherein the GLP-1 receptor agonist is selected from wild-type GLP-1, Exendin-4, GLP-1 variants, and Exendin-4 analogues.
 6. The method of claim 1, wherein the FGF21 receptor agonist is selected from FGF21 variants comprising the following amino acid sequences: (a) (SEQ ID NO: 185) DSSPLLQFGG QVRQRYLYTD DAQETEAHLE IREDGTVGGA AHQSPESLLE LKALKPGVIQ ILGVKTSRFL CQKPDGALYG  SLHFDPEACS FRELLLEDGY NVYQSEAHGL PLHLPGNRSP  HCDPAPQGPA RFLPLPGLPP ALPEPPGILA PQPPDVGSSD  PLAMVGPSQG RSPSYAS; (b)  (SEQ ID NO: 186) DSSPLLQFGG QVRQRYLYTD DAQETEAHLE IREDGTVGGA AHQSPESLLE LKALKPGVIQ ILGVKTSRFL CQKPDGALYG  SLHFDPEACS FRELLLEDGY NVYQSEAHGL PLHLPGNRSP  HCDPAPQGPA RFLPLPGLPP ALPEPPGILA PQPPDVGSSD  PLAMVGGSQG RSPSYAS; (c)  (SEQ ID NO: 187) D SSPLLQFGGQ VRQRYLYTDD ACQTEAHLEI REDGTVGGAA DQSPESLLQL KALKPGVIQI LGVKTSRFLC QRPDGTLYGS  LHFDPEACSF RELLLEDGYN VYQSEAHGLP LHLPCNRSPH  RDPASRGPAR FLPLPGLPPA LPEPPGILAP QPPDVGSSDP  LAMVGGSQAR SPSYAS;  and (d)  (SEQ ID NO: 188) D SSPLLQFGGQ VRQRYLYTDD ACQTEAHLEI REDGTVGGAA DQSPESLLQL KALKPGVIQI LGVKTSRFLC QKPDGALYGS  LHFDPEACSF RELLLEDGYN VYQSEAHGLP LHLPCNRSPH  RDPASRGPAR FLPLPGLPPA LPEPPGILAP QPPDVGSSDP  LAMVGGSQAR SPSYAS.


7. The method of claim 1, wherein the metabolic disorder is obesity.
 8. The method of claim 1, wherein the metabolic disorder is type 2 diabetes mellitus.
 9. The method of claim 1, wherein the metabolic disorder is dyslipidemia.
 10. The method of claim 1, wherein the metabolic disorder is nonalcoholic fatty liver disease (NAFLD).
 11. The method of claim 1, wherein the metabolic disorder is nonalcoholic steatohepatitis (NASH).
 12. The method of claim 1, wherein the metabolic disorder is insulin resistance.
 13. The method of claim 1, wherein the metabolic disorder is hyperinsulinemia.
 14. The method of claim 1, wherein the metabolic disorder is glucose intolerance.
 15. The method of claim 1, wherein the metabolic disorder is hyperglycemia. 